Tunneling nanotubes mediate the transfer of stem cell marker CD133 between hematopoietic progenitor cells

Accepted Manuscript Tunnelling nanotubes mediate the transfer of stem cell marker CD133 between haematopoietic progenitor cells Doreen Reichert, Julia Scheinpflug, Jana Karbanová, Daniel Freund, Martin Bornhäuser, Denis Corbeil PII:

S0301-472X(16)30508-2

DOI:

10.1016/j.exphem.2016.07.006

Reference:

EXPHEM 3438

To appear in:

Experimental Hematology

Received Date: 14 February 2016 Revised Date:

15 July 2016

Accepted Date: 16 July 2016

Please cite this article as: Reichert D, Scheinpflug J, Karbanová J, Freund D, Bornhäuser M, Corbeil D, Tunnelling nanotubes mediate the transfer of stem cell marker CD133 between haematopoietic progenitor cells, Experimental Hematology (2016), doi: 10.1016/j.exphem.2016.07.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Tunnelling nanotubes mediate the transfer of stem cell marker CD133 between haematopoietic progenitor cells

Doreen Reichert1, Julia Scheinpflug1, Jana Karbanová1, Daniel Freund1, Martin Bornhäuser2, 3

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and Denis Corbeil1, 2, *

Tissue Engineering Laboratories, Biotechnology Center (BIOTEC), Technische Universität 2

DFG-Research Center and Cluster of Excellence for

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Dresden, Dresden, Germany,

Regenerative Therapies, Technische Universität Dresden, Dresden, Germany, 3Medical Clinic

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and Polyclinic I, University Hospital Carl Gustav Carus, Dresden, Germany

*Corresponding author: Dr. Denis Corbeil, Ph.D.

Tissue Engineering Laboratories

Biotechnology Center (BIOTEC)

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Technische Universität Dresden

Tatzberg 47-49, 01307 Dresden, Germany Tel./Fax: 49-351-463-40118/244

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E-mail: [email protected].

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Category for the table of content: Microenvironment and Niche

Word count: Abstract and main text (excluding references and figure legend): 7032

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ACCEPTED MANUSCRIPT Abstract

Deciphering all mechanisms of intercellular communication used by haematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated whether these cells can produce thin F-actin-

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based plasma membrane protrusions, referred to as tunnelling nanotubes (TNTs), which are known to bridge cells over long distances without contact with the substratum, and transfer cargo molecules along them in various biological processes. We found that human primary

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CD34+ haematopoietic progenitors and leukaemic KG1a cells develop such structures upon culture on primary mesenchymal stromal cells or specific extracellular matrix-based

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substrata. Time-lapse video microscopy revealed that cell dislodgement is the primary mechanism responsible for the TNT biogenesis. Surprisingly, we found that among various cluster of differentiation (CD) markers only the stem cell antigen CD133 is transferred between cells. It is selectively and directionally transported along the surface of TNTs in small clusters, like cytoplasmic phospho-myosin light chain 2, suggesting that the latter actin

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motor protein might be implicated in this process. Collectively, our data provide new insights into the biology of haematopoietic progenitors, which can help to understand all facets of intercellular communication in the bone marrow microenvironment under healthy or

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cancerous conditions.

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Keywords: CD133, haematopoietic stem cell, intercellular communication, migration, tunnelling nanotube, uropod

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ACCEPTED MANUSCRIPT Introduction

Cell-to-cell communication is a prerequisite for the development and maintenance of multicellular organisms. Various mechanisms for the exchange of molecular information between cells have been documented and one of them relies on the formation of thin plasma

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membrane protrusions, referred to as tunnelling nanotubes (TNTs) [1] (reviewed in Refs [24]). TNTs can bridge cells over significant distances (>100 µm) and might have an underestimated physiological role in communication and signalling during health and disease

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[5-9]. Numerous cargo molecules and organelles such as mitochondria and lipid droplets could be transported along them in various processes including development, immune

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defence, cancer progression and pathogen spreading [3,10,11]. Electric signal could also be transferred between connected cells [12]. TNTs are highly heterogeneous in terms of their connection mode and transferred materials. Although they are subjected to intense research, little is known about them in the context of stem cells [13].

How CD34+ haematopoietic stem and progenitor cells (HSPCs) communicate between

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each other and with their cellular microenvironments nowadays arises as an important question [14,15], as intercellular crosstalk is essential to the maintenance of haematopoiesis and the constant remodelling of the bone marrow niche. The adhesion of HSPCs to

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extracellular matrix or cellular components such as multipotent mesenchymal stromal cells (MSCs) and osteoblasts among others might influence their communication modes [16,17].

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The secretion of molecules and their interaction with receptors on the target cells or contactdependent adhesion/signalling are good examples of mechanisms of intercellular communication. By studying the biology of the stem (cancer stem) cell marker CD133 (prominin-1) [18], our group and others have revealed new facets in the polarization and migration of HSPCs as well as their intercellular communication with their surrounding microenvironment [19,20] (reviewed in Refs [21-23]). For instance, Gillette and colleagues described the selective transfer of membranous components notably CD133 upon physical contact between haematopoietic cells with osteoblasts [24]. The molecular mechanism

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underlying this transfer seems to involve a cytophagocytosis event where a portion of the haematopoietic CD133-containing membrane is engulfed by the osteoblast. In migrating CD34+ HSPCs, CD133 is selectively concentrated in the uropod membrane at the rear pole [19,25]. Another communication mechanism involves small CD133-containing membrane

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vesicles that are specifically released by HSPCs during differentiation process, internalized by MSCs and delivered afterwards to their endosomal compartment [20]. Such phenomenon is in line with exosomes acting as vesicular carriers for intercellular communication.

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Until now, the physiological function of CD133 remains elusive. It was proposed that this cholesterol-binding glycoprotein is involved in the remodelling of plasma membrane,

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particularly membrane protrusions [26,27]. Its association with specific membrane microdomains (often referred to as membrane rafts) [26,28] and its interaction with the phosphoinositide 3-kinase (PI3K) are consistent with such role [29]. In human haematopoietic system, CD133 highlights subsets of CD34+ progenitor cells, particularly lympho-myeloid ones, while the CD34+CD133–/low phenotype labels erythro-myeloid progenitors [30-32]. In

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cancer, CD133 is widely used to isolate cancer stem cells (reviewed in Ref. [33]). Here, we demonstrate that CD34+ HSPCs and KG1a cells, an acute myelogenous leukaemia cell line that cannot differentiate into mature cells and remains in an early state of

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development [34,35], develop TNTs that link adjacent cells. Our analyses reveal that i) TNT biogenesis relies on cell migration, which in turn can be influenced by the supporting

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substratum; ii) TNTs are formed from uropod membrane; iii) CD34+CD133high and CD34+CD133–/low HSPC subpopulations display different propensity to generate TNTs, and iv) a selective transfer of CD133 occurs via TNTs, which reveals a new mechanism of intercellular communication used by CD34+ haematopoietic progenitors and leukaemic cells.

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ACCEPTED MANUSCRIPT Methods HSPC and MSC isolation, cell culture

Human HSPCs were collected from healthy donors after informed consent and approval of the local ethics committee (Ethikkommission an der Technischen Universität Dresden (TUD),

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Ethic board no. EK201092004) and isolated from mobilized peripheral blood directly after leukapheresis by magnetic-activated cell sorting (MACS) technology based on CD34 or CD133 (Miltenyi Biotec) as described [36,37]. The age of the donors (n = 21, 57% female)

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ranged from 20 to 53 years (average 31). CD34+ or CD133+ HSPCs were cultured in HSPC medium [serum-free medium (CellGro SCGM, CellGenix) supplemented with early-acting

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cytokines (50 ng/ml stem cell factor, 50 ng/ml fms-related tyrosine kinase-3 ligand (CellGenix) and 15 ng/ml interleukin-3 (R&D Systems)] at a density of 7.5 x 104 per cm2 of surface area for two to three days on MSCs in a humidified 5% CO2 atmosphere at 37ºC [37]. Afterward, they were collected, labelled with different dyes (see below) and cultured on different substrata.

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Primary MSCs were extracted from bone marrow aspirates collected from healthy donors after informed consent. The study was approved by the local ethics committee (Ethikkommission an der TUD, Ethic board no. EK263122004). The age of the donors (n = 3)

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ranged from 25 to 35 years [37]. Plastic-adherent MSCs were obtained and cultured in MSC medium [DMEM, low glucose and 2 mM L-glutamine (#31885-023, Thermo Fisher

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Scientific)] supplemented with 10% fetal calf serum (FCS; PAA, GE Healthcare) as described [36,37]. They were used between passages 3 and 5. The CD34+ acute myelogenous leukaemia cell line KG1a (ACC 421, German Collection

of Microorganisms and Cell Cultures (DSMZ)) was cultured on different substrata in RPMI 1640 with 10% FCS at 37°C in a humidified 5% CO2 atmosphere. We used either this medium or serum-free HSPC medium when they were co-cultured with CD34+ HSPCs. Both conditions gave the same results (data not shown).

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ACCEPTED MANUSCRIPT Reagents

The ROCK inhibitor Y-27632 (#688001) and latrunculin B (#428020) were obtained from Calbiochem and Merck GmbH, respectively. Nocodazole (#M1404), methyl-β-cyclodextrin (MβCD; #C4555), 4,6-diamidino-2-phenylindole (DAPI; #32670), chicken collagen II

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(#C9301), human collagen IV (#C5533), gelatin (#G1393), hyaluronic acid (#53747), and poly-L-lysine solution (#P8920) were purchased from Sigma-Aldrich. Fibronectin (#356008) and laminin (#354232) from BD Biosciences. Collagen type I (Collagen R Solution 0,2%;

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#47254) was ordered from Serva, stromal cell-derived factor-1α (SDF-1α) from Strathmann GmbH & Co. KG. VybrantTM Multicolor Cell-Labeling Kit (#V-22889), green fluorescent

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CFDA-SE (carboxyfluorescein diacetate succinimidyl ester, #C1157) and Alexa Fluor® 488conjugated phalloidin (#A12379) from Thermo Fisher Scientific.

Flow cytometry

Isolated HSPCs were resuspended in PBS containing 1% bovine serum albumin and aliquots

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of 100,000 cells /100 µl were incubated with allophycocyanin (APC)-conjugated CD34 (clone 581, #555824, BD Biosciences) and phycoerythrin (PE)-conjugated CD133 (clone AC133; #130-080-801, Miltenyi Biotec) antibodies or the corresponding isotype controls for

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30 min at 4°C. After washing with PBS, 10,000 events were acquired on an LSRII flow cytometer (BD Biosciences). Instrument settings and gating strategies were established using

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isotype controls, and data were analysed with FlowJo software (TreeStar, Ashland, Oregon, USA). The median fluorescence intensity (MFI) value of the isotype control antibody was subtracted from the MFI relative to CD133 antigen.

Plasmid construction and transfection Human CD44.1 [GenBank accession number AY101193], CD53 [BC040693], CD63 [BC002349] and CD133 [AF027208] cDNAs were cloned either into pEGFP (enhanced green fluorescent protein)-N1 or pmCherry-N1 vector (Clontech Laboratories Inc). Selective

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PCR amplifications introducing unique restriction enzyme sites were performed using the following oligonucleotides: CD44.1: 5’-CGCCTCGAGATCCTCCAGCTCCTTT-3’ and 5’ATGGTGTAGAATTCGCACCCCAATC-3’;

CD53:

5’-

GTGCCTCGAGAAGGGCAAGAATAT-3’ and 5’-CTGCAAGCTTTAGCCCTATGGTCTCD63:

5’-GAACCTCGAGCCAGCCTTGGGAAG-3’

CAGAAAGCTTCATCACCTCGTAGC-3’;

and

TTGGAGTTTCTCGAGCTATGGCCCTCGTACT-3’

and

5’-

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3’;

CD133:

5’-

and

5’-

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TTCAACATCAGCTCGAGATGTTGTGATGG-3’ as 5’ and 3’ primers, respectively. The resulting PCR fragments were digested with XhoI/EcoRI (CD44.1), XhoI/HindIII (CD53,

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CD63) or XhoI (CD133), respectively, and cloned into the corresponding site(s) of opened vector. CD34-GFP was purchased from ORIGENE (#RG204446, vector: pCMV6-AC-GFP; human CD34, NM_001025109.1).

KG1a cells (2 x 106 cells in 100 µl) were transfected by electroporation with 4-8 µg of plasmid DNA using AmaxaTM NucleofectorTM kit L according to the manufacturer’s

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instructions (#VVCA-1005, Lonza). After transfection, cells were washed in pre-warmed medium and spun down, and cell pellets were incubated for 15 min at 37ºC in a humidified 5% CO2 atmosphere [38]. Cells were resuspended in 1 ml pre-warmed medium, seeded onto

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35-mm dishes, and incubated overnight at 37°C prior usage.

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Surface coating and chemical treatments Glass coverslips were coated with either human fibronectin (5 µg/cm2 of surface area), collagen type I (160 µg/cm2), collagen II (10 µg/cm2), collagen IV (10 µg/cm2), gelatin (200 µg/cm2), poly-L-lysine solution (10 µg/cm2), hyaluronic acid (50 µg/cm2) or laminin (5 µg/cm2) according to the manufacturer’s procedures. Cells were incubated in a chamber with 5% CO2 atmosphere at 37ºC for an indicated given time with 1 mM MβCD, 10 µM ROCK inhibitor Y-27632, 100 ng/ml SDF-1α, various concentrations of nocodazole (0.5 and

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1 µM) or latrunculin B (0.1 to 5 µM) directly after seeding or 2 hrs upon culture as indicated. The appropriate vehicle controls were used.

Dye labelling and live-cell imaging

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Cells were labelled with VybrantTM cell-labelling solutions or CFDA-SE dye. In brief, 1 x 106 cells were gently resuspended in 1 ml serum-free media and labelled with 1 µM 3,3’dioctadecyloxacarbocyanine

perchlorate

(DiO)

or

1,1-dioctadecyl-3,3,3,3-

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tetramethylindocarbocyanine perchlorate (DiI) for 30 min or 1 µM CFDA-SE for 15 min at 37°C. After labelling, cells were washed thrice with pre-warmed RPMI 1640 supplemented

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with 10% FCS (for KG1a cells) or MACS-buffer (PBS containing 0.5 mM EDTA and 0.5% human serum albumin; for HSPCs). Cells were seeded at a density of 8 x 104/cm2 of surface area on fibronectin-coated glass coverslips in silicon eight-well chambers (flexiPerm, Greiner Bio-One GmbH) and cultured in the appropriate medium. Images were captured at 37°C under 5% CO2 atmosphere using a Leica TCS SP5 inverse confocal laser-scanning

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microscope (CLSM) equipped with a resonant scanner for high-speed imaging, a Leica Hybrid Detector and a 63.0 x 1.2 water UV objective. To quantify the number of TNTs, we took pictures either randomly (KG1a cells) or systematically throughout coverslip (CD34+

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HSPCs), and then counted the number of TNTs associated with a given number of cells.

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Cell surface and intracellular labelling Cell surface labelling was performed by adding APC-coupled CD133 antibody (#130-090826, Miltenyi Biotec) and/or fluorescein-labelled wheat germ agglutinin (WGA; Vector Laboratories) to the culture media for 15 min at room temperature, and living cells were directly imaged as described above. For the intracellular labelling, cells were fixed with 4% paraformaldehyde (PFA) for 30 min, quenched with 50 mM NH4Cl for 10 min and permeabilized with 0.2% saponin prior the immunolabelling with rat anti-α-tubulin antibody (#MCA78S, AbD Serotec) or rabbit anti-phospho-myosin light chain (MLC) 2 (Thr18/Ser19)

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antibody (#3674, Cell Signaling Technology) for 30 min at room temperature followed by Cy3® goat anti-rat and Cy3® goat anti-rabbit secondary antibody (both from Jackson ImmunoResearch), respectively. The F-actin in TNTs of DiI-labelled cells was visualized using Alexa Fluor® 488-conjugated phalloidin. Nuclei were labelled with DAPI (1 µg/ml).

UV objective without mounting the samples.

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Time-lapse video microscopy

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Images were captured using a Zeiss LSM700 inverse CLSM equipped with a 63.0 x 1.40 oil

Cells were seeded on different substratum-coated glass coverslips attached to a silicone eight-

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well chamber and cultured for 2 hrs. During the time-lapse recording, cells were kept in a chamber with 5% CO2 atmosphere at 37ºC. To analyse migration behaviour, serial images were captured at 30-sec intervals with an inverted Leica AF6000 LX microscope equipped with a 20.0 x 0.70 Dry (for cell tracking) and 40.0 x 1.25 oil objective (for visualization of TNTs), respectively. For immunofluorescence analysis, serial images were taken at 1- or 30-

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sec intervals with an inverted Leica TCS SP5 inverse CLSM equipped with a resonant scanner, a Leica hybrid detector and a 63.0 x 1.2 water UV objective.

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Scanning electron microscopy

Cells were grown on fibronectin-coated coverslips or co-cultured on MSCs. Cells were fixed

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in 2% glutaraldehyde for 1 hr at room temperature and then overnight at 4°C. After being subjected to dehydration in an acetone gradient (25–100%), cells were critical point-dried in a CO2 system (Leica EM CPD300 automated Critical-point dryer). Samples were then sputtercoated with gold (Sputter Coating Device SCD 050, BAL-TEC GmbH) and examined at 5-kV accelerating voltage in an environmental scanning electron microscope (JSM-7500F, Jeol).

Image and statistical analyses The 2- and 3-dimensional images were analysed using ImageJ [39] and Volocity 6.3

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(PerkinElmer), respectively. Final images were prepared with Adobe Photoshop and Adobe Illustrator software (Adobe). ImageJ was used for the manual tracking of cells obtained from video microscopy. Data are expressed as the mean ± standard deviation of at least three independent

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experiments except for the SDF-1α ones where the number of individual cells analysed was higher. Box and-whisker plots represent 50% of the values within the box and minimum and maximum of all of the data within the whiskers. Horizontal lines within the box represent

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median values. Statistical analyses were performed using two-tailed paired or unpaired student’s t-test with GraphPad Prism 6 (GraphPad Software Inc.). Differences were regarded

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p < 0.001;**** p < 0.0001.

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as significant if the calculated two-tailed p-values were < 0.05. *, p < 0.05; **, p < 0.01; ***,

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ACCEPTED MANUSCRIPT Results Visualization of tunnelling nanotubes

To identify and characterize TNTs produced by haematopoietic cells we labelled human primary

CD34+

HSPCs

isolated

from

leukapheresis

by

MACS

or

leukaemic,

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immature/undifferentiated, haematopoietic KG1a cells with fluorescent membrane dyes DiO (green) or DiI (red), mixed and incubated them 2 hrs on fibronectin-coated support prior to live-cell analysis by CLSM. As illustrated in figure 1, both cell types developed narrow

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membrane processes that were not in contact with the substratum, the hallmark of TNTs. Substrate-adherent protrusions such as filopodia were also observed (see below Fig. 3D, E).

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Given that TNTs are noted between differentially (DiO/DiI) labelled cells we could exclude that they are remnants of cell division. CD34+ HSPCs displayed a limited number of TNTs. Just 0.1% (i.e. 0.06 ± 0.02%) of them harboured TNTs (Fig. 1A-C, 80000 cells were analysed per experiment, n = 4 independent donors). In contrast, 13.97 ± 3.31 TNTs were generated per 100 KG1a cells (Fig. 1D-E, ≈300 cells per experiment; n = 7). While ≈95% of TNTs

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interconnected 2 cells, ≈5% of them were formed between 3 cells (Fig. 1C, see below Fig. 4A). 82.70 ± 4.92% of TNTs were longer than 5 µm, and could extend over more than 50 µm (Fig. 1). The number of TNT did not increase after 5 hrs of incubation (data not shown).

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Interestingly, TNTs observed between two CD34+ HSPCs were in all cases derived from only one cell (Fig. 1A, B), whereas about half (44.6 ± 8.1%) of those connecting two KG1a

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cells originated from both (Fig. 1D, E). Throughout our investigation, we have detected only one TNT from CD34+ HSPCs with a dual colour, and it involved a TNT network (Fig. 1C). The contact zone between TNT-membranes produced by CD34+ HSPCs or KG1a cells also differs. The TNT/cell body membranes contact generated by CD34+ HSPCs appeared tight (Fig. 1A-C, yellow arrowhead) while KG1a cell-derived TNTs did not seamlessly mix (Fig. 1D, E, green arrowhead). The latter TNTs were similar to those produced by T cells [5]. In all cases, the lack of diffusion of membrane dyes between cells suggests that TNTs contain a junctional complex (see below). To rule out any artefact originating from culture conditions,

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CD34+ HSPCs and KG1a cells were differentially labelled and incubated together (Fig. 1F, G). In the co-culture system, all TNTs were extending from the KG1a cells, a phenomenon that is consistent with their propensity to generate them. The TNT contact zones that linked CD34+ HSPC and KG1a cells were also reminiscent of those produced by KG1a cells (Fig.

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1F, G, green arrowhead). The reason behind these singularities remains to be identified, but it might reflect the origin of cells (healthy versus cancerous; see Discussion).

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Ultrastructure of tunnelling nanotubes

To gain insights into the TNT ultrastructure we performed a scanning electron microscopy

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(SEM) analysis. Although membrane bulges were observed along the TNTs by CLSM and SEM analyses in both cell types (Fig. 2A, B, D, red circle, see also Fig. 3D, control), their overall thickness appeared to be smaller than 100 nm in diameter (Fig. 2B, C). In agreement with fluorescent images, we observed an apparent membrane continuity between TNT ending points and the CD34+ HSPC body (Fig. 2B, yellow arrowhead) while a clear junction within

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TNT membrane was detected in KG1a cells (Fig. 2C, D, green arrowhead). The latter observation being reminiscent to the dual DiO-/DiI-labelled TNT membranes generated by

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KG1a cells (Fig. 1E).

Impact of F-actin on the structure of tunnelling nanotubes

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We investigated the structural components of TNTs. We concentrated our efforts on TNTs generated by KG1a cells given their number facilitates a quantitative analysis. We found that haematopoietic cell-derived TNTs contained F-actin as observed with fluorochromeconjugated phalloidin labelling (Fig. 3A, arrowhead). The importance of F-actin for their formation and/or stabilization is demonstrated by the observation of the effect latrunculin B, a drug that prevents the actin assembly. When DiO-/DiI-labelled cells were incubated directly after seeding with different latrunculin B concentrations for 2 hrs, a significant reduction of the TNTs was already detected at a low drug concentration (0.5 µM) (Fig. 3C). Only

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0.62 ± 0.68 TNTs per 100 cells were found by comparison to 12.50 ± 2.50 TNTs without drug addition (Fig. 3C, D, control, p ≤ 0.001). No TNT was detected at 5 µM latrunculin B (Fig. 3C, D, imaging I). Latrunculin B affected not only the formation of TNTs, but also

evidenced after drug removal (Fig. 3D, imaging II).

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destabilized existing ones (Fig. 3D, imaging III), and its negative impact was reversible, as

Given that certain TNTs, particularly thicker ones, were reported to contain microtubules [40,41] we investigated the presence of α-tubulin. We didn’t detect α-tubulin within TNTs

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upon co-staining with phalloidin (Fig. 3B). Moreover, the incubation of cells with up to 1 µM nocodazole, a concentration that leads to the depolymerization of microtubules in

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haematopoietic cells [19,20], had no effect on the formation of TNTs (Fig. 3B, C).

Impact of membrane cholesterol on the structure of tunnelling nanotubes To determine whether membrane cholesterol plays a structural role in TNT, DiO-/DiI-labelled KG1a cells were incubated with 1 mM MβCD for 10 min up to 60 min prior to live-cell

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imaging. The MβCD is known to deplete cholesterol from biological membranes [42]. Surprisingly, a substantial reduction of the number of TNTs was observed only after 1 hr incubation (Fig. 3E, F; 53.80 ± 15.11%, p = 0.00254). Under these conditions, the number of

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cells without podia was significantly elevated (Fig. 3F, 43.81 ± 9.23%). At higher MβCD

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concentration, i.e. 5-10 mM, all cell rounded up (data not shown) [24].

Biogenesis of tunnelling nanotubes relies on adhesion and cell migration: Implication of the uropod

To decipher the mechanism underlying the TNT formation, we evaluated the influence of the supporting matrix. Interestingly, DiO-/DiI-labelled CD34+ HSPCs and KG1a cells developed TNTs when they are cultured on fibronectin or MSCs used as a feeder cell layer (Fig. 4A), but not on other substrata (Fig. 4B, C). For instance, CD34+ HSPCs growing on collagens (I, II, IV), laminin, hyaluronic acid, poly-L-lysine and gelatin generated very rare and short

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TNTs (Fig. 4B; data not shown). In contrast, KG1a cells did not firmly adhere to other substrata with the exception of poly-L-lysine. No TNT was observed on the latter support (Fig. 4C; data not shown). Thus, the propensity of haematopoietic progenitors to develop TNTs seems to be dependent on the extracellular matrix-based substrata. The tracking of

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individual unlabelled CD34+ HSPCs and KG1a cells from 30 min period–time-lapse movies revealed an intriguing correspondence between TNT formation and cell migration (Fig. 4A-C, 50 cells per condition; three independent donors were used for the CD34+ HSPCs). TNTs

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were detected when a significant cell migration was observed (i.e. on fibronectin and MSCs) (Fig. 4A). When the distance of cell migration was reduced by about half, e.g., CD34+ HSPCs

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growing on collagen I, or KG1a cells on poly-L-lysine, rather than MSCs as a substrate, almost no TNT was recorded (Fig. 4B and C, respectively). Of note, we could observe under these conditions the presence of highly motile plasma membrane protrusions, referred to as cytonemes/magnupods. These structures never came in firm contact with another cell to form a stable TNT (Fig. 4B-D).

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One essential characteristic of migrating haematopoietic progenitors is the acquisition of a polarized morphology, which can include the formation of a uropod at the rear pole [19]. Surprisingly, TNTs are often linked to this structure as observed by various microscopy

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techniques (Fig. 5A). Time-lapse video microscopy demonstrated the generation of a TNT from the uropod membranes when one of two contacted cells is moving apart (Fig. 5B,

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Supplementary Video S1). The functional link between migration and TNT formation was provided by the incubation of KG1a cells with the ROCK inhibitor Y-27632, which inhibits the uropod formation and cell migration [19]. Cells incubated with latrunculin B were used as a positive control. Latrunculin B prevents not only TNT formation but also cell migration [19]. The tracking diagrams of individual KG1a cells cultured under these conditions revealed a direct relationship between migration (Fig. 5C, D, 50 cells per condition, n = 3) and the presence of TNTs (Fig. 5E). Indeed, very few TNTs were observed when cell migration was significantly impaired.

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The addition of the chemokine SDF-1α,  chemoattractant known to promote the directional cell migration in transwell assays [19,20], had no additional effect on the migration of KG1 cells and TNT formation (Supplementary Fig. S1A-D). When a similar experiment was performed with CD34+ HSPCs the number of TNTs was neither increased

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nor reduced (Supplementary Fig. S1E-G; see Discussion), although we observed that the cellular movement of a given migrating cell was further augmented (from 75 ± 32 to 99 ± 41 µm per 30 min).

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All together, these data suggest that the mechanism responsible for TNT biogenesis relies primarily on cell migration, the uropod membrane and a direct contact of two cells, which in

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turn can be influenced by the supporting substratum.

The analyses of different haematopoietic progenitors reveal a link between tunnelling nanotube formation and cell morphology

To get more insight into the propensity of primary CD34+ HSPCs to produce TNTs, we

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isolated two distinct subpopulations, CD34+CD133high and CD34+CD133–/low cells. The CD34+ subpopulation harbouring CD133 were isolated by MACS using CD133 antigen and CD34+CD133–/low HSPCs were collected from the flow-through and enriched by means of

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CD34 selection (Fig. 6A). Flow cytometry analyses revealed that >95% of cells derived from the first selection (referred to as CD133-MACS) were CD34+CD133high, whereas CD34+ cells

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collected from flow-through (CD133-FT) contained ≈80% and <20% of them with CD34+CD133– or CD34+CD133low phenotype, respectively (Fig. 6A), as described previously [30].

To evaluate the potential of CD133-MACS and CD133-FT HSPC subpopulations to

produce TNTs, cells were labelled with membrane dye and incubated 2 hrs on fibronectincoated support prior to live-cell analysis by CLSM. Although the live-cell analysis revealed that they generated very few TNTs by comparison to the KG1a cells as described above, DiOlabelled CD133-MACS cells nonetheless developed more than twice the amount of TNTs

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compared to CD133-FT cells suggesting that the biological characteristics of these cell subpopulations might be distinct (Fig. 6B, 80000 cells were analysed per experiment, n = 3 independent donors). In light of the data presented in the previous section, this new observation prompted us to explore in more details the migration and morphology of these

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distinct CD34+ HSPC subpopulations. The tracking of individual unlabelled CD133-MACS HSPCs cultured on MSCs in 30 min period–time-lapse movies revealed a pattern of migration similar to that of CD34-selected HSPCs (Fig. 6C), which is consistent with the fact that a

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large fraction of cells are double positive for CD34 and CD133 [30]. In contrast, CD133-FT cells (i.e. CD34+CD133–/low) exhibited a greater migration distance than CD133-MACS cells

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under the same conditions (Fig. 6C). The bright-field video recording revealed that numerous migrating cells within the CD133-FT fraction maintained a spherical morphology in contrast to elongated cells found in CD133-MACS subpopulation (Fig. 6D, left and middle panels). When the backward structure of an elongated cell came into contact with MSC, the cell stopped to migrate and started to rotate – a process that reduces its migration distance within a

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given time (Supplementary Video S2). The quantification indicates that cells with an elongated morphology represented 85.15 ± 3.51% and 24.94 ± 3.11% in CD133-MACS and CD133-FT cell fractions, respectively (Fig. 6D, right panel). Unexpectedly, the ultrastructure

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analysis uncovered that CD34+CD133high and CD34+CD133–/low cells are morphologically distinct (Fig. 6E). Consistent with the formation of TNTs and cell migration, almost all cells

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in the CD133-MACS fraction contained a uropod at the rear pole (Fig. 6E, green asterisk), while those in CD133-FT fraction did not. Migrating cells either harboured a small uropodlike structure at the apex of their rounded cell body or were devoid of such appendage (Fig. 6E, red asterisk and star, respectively). A lamellipodium was observed at the leading edge of all migrating cells (Fig. 6E). Thus, the characterization of different CD34+ HSPC subpopulations demonstrates again the connexion between TNT biogenesis and the uropod structure. The physiological relevance of such differential biological activities between subpopulations of haematopoietic progenitors remains to be determined.

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Tunnelling nanotubes are not open-ended The data acquired using fluorescent membrane probes suggest the absence of a direct continuity of the membranes of the connected cells (Fig. 1). The lack of membrane mixing

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could be explained by the presence of a junction between them as reported in other cellular systems [5]. In line with this hypothesis, we observed that with CFSE covalently labelled long-lived intracellular molecules did not flow seamlessly between cells although they did

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enter TNTs indicating that the latter are not open-ended tunnels (Fig. 7). Although membrane or cytosolic components did not diffuse passively between the connected cells, we could not

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exclude that specific [or particular] proteins may be transferred. Of note, we observed membrane fragments that were attached to the opposite cells suggesting the breakage of TNTs as an alternative mechanism of intercellular communication (Supplementary Fig. S2).

Intercellular transfer of CD133-GFP through tunnelling nanotubes

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To investigate whether particular proteins can use a TNT as a communication device, we engineered a selected set of membrane proteins with GFP and/or mCherry technologies notably i) stem cell markers CD34 and CD133; ii) two tetraspanin membrane glycoproteins

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CD53 and CD63 (lamp-3) that are often associated with intracellular vesicular-like structures, and iii) CD44, a transmembrane glycoprotein involved in cell-adhesion [25]. Upon

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transfection of KG1a cells, all engineered CD markers were observed in TNTs (Fig. 8). CD34/CD44/CD53/CD63 fusion proteins displayed a continuous labelling along TNTs while CD133-mCherry (or GFP version) showed punctate signals (Figs 8A, 9A, B, white arrows). Not all TNTs showed a CD133 labelling indicating that the sole expression of CD133 is not a prerequisite for its localization in TNT (Fig. 8B). A faint and more continuous CD133-GFP signal was also observed in TNT when a transfected cell strongly expressed it (Fig. 9A, B, bracket, cell I). Intriguingly, the second TNT-connected cell contained solely a minute CD133-GFP signal suggesting the intercellular transfer of CD133 from one cell to the other

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(Fig. 9A, B, red arrows, cell II). In contrast, no GFP signal was observed in the second cell with the other CD markers (Fig. 9E). These data prompted us to monitor in live the potential exchange of CD133 between cells. Time-lapse video microscopy revealed that the punctate CD133-GFP signals all along the TNTs were moving in a directional fashion from cell I to

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cell II (Fig. 9C, D, Supplementary Video S3). We did not observe a retrograde transport of the CD133-GFP to cell I once transferred to cell II. Yet, we detected the formation of a CD133-GFP+ bulge within the TNT that seemingly did not move (Fig. 9D, red dashed line,

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Supplementary Video S4). Such local CD133-GFP accumulation might correspond to a junctional complex noted between dual DiO-/DiI-labelled TNT membranes (Fig. 9D, last

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panel). These membrane borders within a TNT were also visualized with CD44-mCherry (Fig. 8C). When similar experiments were performed with CD34+ HSPCs, we detected only very weak signals of the transgenes, which impeded their monitoring (data not shown).

Transfer of CD133-GFP occurs via the cell surface

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The TNT-mediated transfer of cargo molecules could occur at the plasma membrane or within TNT via cytoplasmic structures. The dual localization of CD133 at the plasma membrane and endosomal compartment of haematopoietic cells raises the possibility that its

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transfer may operate by one, or both mechanisms [20,43]. To address this issue, we have added fluorochrome-coupled anti-CD133 antibody to living CD133-GFP transfected KG1a

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cells in order to catch a potential fraction of CD133-GFP at the cell surface. Interestingly, only the punctate CD133-GFP signals along TNTs were detected with anti-CD133 antibody indicating that its transfer occurs mainly via the cell surface (Fig. 10A). TNTs produced by CD34+ HSPCs also displayed both CD133 punctate labelling and surface localization (Fig. 10B). Until now, a limited number of mechanisms have been described to explain the active transfer of cargo molecules along TNTs. One of them relies on the acto-myosin machinery [44]. Therefore, we investigated the presence of the regulatory phospho-MLC2, which is expressed by haematopoietic cells [45,46]. Excitingly, MLC2 was detected in TNTs, and

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appeared as a punctate staining similar to CD133 suggesting a potential implication of this

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myosin in TNT-mediated intercellular transfer of cargo proteins (Fig. 10C).

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ACCEPTED MANUSCRIPT Discussion

We report four novel observations concerning the biology of CD34+ HSPCs and leukaemic cells. First, they generate TNTs that establish intercellular contacts in long distances. Second, the biogenesis of TNTs is dependent on cell migration and a direct contact of two (or more)

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cells. Third, the membrane of the backward structure (uropod) of migrating cells is involved in TNT formation. Fourth, the stem cell marker CD133 is selectively transported along the TNTs and exchanged between the connected cells.

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An important aspect of the cellular biology of haematopoietic progenitors is to understand how they can communicate with their cellular microenvironment known as the stem cell

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niche. Here we observed that haematopoietic progenitors and leukaemic cells develop thin, actin-dependent plasma membrane protrusions that link adjacent haematopoietic cells. The structure of TNTs differs from other plasma membrane protrusions such as filopodia by the fact that they are not in direct contact with the substratum [36,47]. Substrate-adherent membrane projections might act as searching-sensor processes to guide haematopoietic

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progenitors to an appropriate microenvironment but they have not been described to be involved in intercellular communication as TNTs [36,47-50]. Two models have been described to explain TNT biogenesis [51]. The first one relies on

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the formation of a motile actin-driven membrane protrusion that is directed towards a target

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cell. We could observe such cytoneme/magnupod-related structures emerging from haematopoietic cells but none making a direct contact with another cell. The second model involves cell motility. Here we found that cell migration was required for TNT formation. We indeed noted that specific substrata that impede either cellular attachment or migration also blocked the generation of TNTs, suggesting that TNT biogenesis can be achieved within the bone marrow only when haematopoietic progenitors are interacting with proper matrix molecules and/or surrounding stromal cells and migrating. A direct contact between two haematopoietic cells is essential to the generation of TNTs, but the number of TNTs would not necessarily increase with a longer distance of cell migration as observed upon the addition

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of SDF-1α to CD34+ HSPCs or with different HSPC fractions. Actually, one could expect a reduction of TNTs when cells are migrating too far away from each other. The membrane fragments observed on opposite cells might reflect the breakage of TNTs upon an extended migration distance of TNT-connected cells (Fig. S2). Microcavities within 3-dimensional

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microenvironment of bone marrow niches might timely and spatially regulate the cell migration distance and consequently the formation of TNTs and their maintenance. Such information is particularly relevant in the context of the development of fully functional

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engineered stem cell niches [52]. The next challenge is now to characterize TNTs generated by haematopoietic cells in their proper niches (i.e. in vivo). New imaging techniques, such as

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two-photon laser-scanning intravital microscopy, might help to achieve this goal [53]. The fact that TNTs are generated preferentially from the uropod membrane suggests a new function of this organelle-like structure that is known to be involved in intercellular adhesion, cell motility and migration [25,54]. The surprising difference in capacity to produce TNTs between CD34+CD133high and CD34+CD133–/low cell subpopulations as well as their

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differential motility behaviour illustrate the importance of this backward structure in these processes. Why CD34+CD133–/low cells have no uropod or harbour only a small one at the apex of their spherical cell body is still an open question. Specific cytoskeleton elements and

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cell surface receptors and/or adhesion molecules are found therein, notably CD133 in CD34+ HSPCs [25,54]. The segregation of adhesion proteins from the backward to the zenith of the

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cell body can explain both phenomena, i.e. the increased migration distance and the reduced number of TNTs produced by CD34+CD133–/low cells, given that their uropod will not engage physical contact with the substratum and adjacent cells, respectively. Is CD133 involved in uropod formation? The RNA interference-mediated knockdown of CD133 in CD34+ HSPCs has shown that CD133 is dispensable for uropod formation, elongated morphology and migration [55]. It remains to be evaluated whether the expression of proteins that connect actin to the plasma membrane cell cortex differ between HSPC subpopulations. The ezrinradixin-moesin (ERM) proteins are good candidates to be investigated. The lipid composition

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of the uropod also differs from that of the other plasma membrane subdomains of migrating haematopoietic progenitors (e.g., lamellipodium at the leading edge) [24,25] (reviewed in Refs [22,56]). Thus, specific membrane and cytoplasmic peripheral proteins, lipids and the underlying cytoskeleton may thus make the uropod more prone to deformation. Observed in

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neutrophils, monocytes and natural killer cells, uropods are also involved in complex interplay between immune cells [56,57]. In cathepsin X-upregulated T lymphocytes exhibiting persistent lymphocyte function-associated antigen 1 (LFA-1) integrin activation,

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tube-like structures are formed by extended uropods that subsequently elongate [58], which might represent the final step in leukocyte extravasation through inflamed vessels [59]. These

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processes are nonetheless different from the nanoscale-radius tube observed here where the general structure of the uropod remains intact.

Mediating the transfer of biological information between connected cells in a coordinated manner could be an asset of TNTs, i.e. the recipient cell could be selectively targeted, which is not necessarily the case when small membrane vesicles are used as signalling devices [60].

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Here, we observed a selective transfer of CD133 along the plasma membrane of TNTs. Surprisingly, this phenomenon was not observed for other CD markers. The biological characteristics of the pentaspan membrane glycoprotein CD133, i.e. i) its direct interaction

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with membrane cholesterol, ii) its association with specific membrane rafts where sterol molecules in concert with longer-chain saturated phospholipids form condensed molecular

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complexes that promote fluid phase separations within the lipid bilayer [61], and iii) its concentration in various plasma membrane protrusions including those in haematopoietic cells, might explain such uniqueness [20,26,28,62,63]. The tight clustering of membrane cholesterol–CD133 complexes along TNTs could be part of the mechanism underlying its selective transfer. Indeed, it is extremely surprising to observe that about half of all TNTs, which are not stabilized by interaction with the substratum, remain intact upon MβCD treatment (1 mM, 1-hr exposure). A similar observation has previously been made using another cell type (i.e. malignant urothelial cancer cell line) where a significant reduction of

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TNTs was detected solely after a prolonged treatment (2 hrs) with higher MβCD concentrations (4-6 mM) [64]. These data suggest that either the amount of cholesterol in TNTs is very low or cholesterol therein is strongly compacted with other lipids and/or scaffolding proteins, and hence inaccessible to MβCD. In both scenarios, the lipid

67], but also by selecting the transfer of membrane cargos [68].

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components of TNTs could play a role not only by generating a highly curved membrane [65-

The tight interaction of lipid–lipid and lipid–protein complexes with the actin cytoskeleton

docking

phospholipids

such

as

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should not be neglected. Indeed, the inner leaflet of membrane rafts is often enriched with phosphatidylinositol(4,5)-bisphosphate

(PIP2)

and

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phosphatidylinositol(3,4,5)-triphosphate (PIP3) that regulate the interplay between plasma membrane and actin cytoskeleton [69]. Wang and colleagues have observed the potential involvement of PIP3 in TNT formation by inhibiting the PI3K activity in astrocytes [70], while Gousset and colleagues reported that a point mutation in the molecular motor myosin-X affecting its binding to PIP3 impaired the induction of TNTs in neuronal cells [71]. Myosin

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Va has been also reported in TNTs [1]. In this context, the punctate staining of regulatory phospho-MLC2 along TNTs (just like CD133) is significant. A potential interaction of the actin motor protein myosin with membrane cholesterol–CD133 complexes might explain the

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selective movement of CD133 at the TNT surface. Myosins have been found in membrane rafts [72,73]. It might be more than a coincidence that the phosphorylation of CD133 by Src

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and Fyn tyrosine kinases promotes its interaction with the regulatory subunit (p85) of PI3K [29]. Thus, an orchestrated mechanism involving membrane rafts (including their cargo proteins), an enzymatic transformation of local docking phospholipids (e.g., PIP2
PIP3) and myosin motor proteins might regulate the intercellular transport of particular membrane proteins (Fig. 11). It remains to be determined how such protein–lipid complexes can cross the TNT-associated junctional complex generated by two connected CD34+ cells. Various models have been proposed to explain the presence of a membrane border in TNTs including the presence of gap junctions that select and/or exclude the transfer of certain

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cytoplasmic and membrane components or organelles [12]. The phagocytosis of the TNTending point by the recipient cell is another potential mechanism of transfer that can also explain the lack of retrograde transport of CD133 between the TNT-connected cells. Irrespectively of the mechanism regulating the intercellular transfer of CD133, it is important

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to note that HIV-1 transmission between TNT-connected T cells occurs in a very similar way indicating that the current observation is not unique to CD133 [5]. Although, we did not observe the transfer of organelles (e.g., mitochondria using MitoTracker Red, data not shown)

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between haematopoietic progenitors we cannot exclude that such event may occur between haematopoietic cells and MSCs as recently demonstrated for cancer cells [74,75]. The

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presence of membrane bulges along the TNTs suggests transfer of organelles. Obviously, the nature of the cellular compounds transferred by TNTs varies considerably between cell types as well as the molecular machinery underlying the cargo transfer [10,51]. Further investigation is required to unravel all biological aspects of TNTs. In particular, it will be of interest to explain the morphological differences observed between TNTs generated by

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CD34+ HSPCs and KG1a cells particularly at their contact zones as well as the difference in the propensity of these progenitors to produce them. Nonetheless, the low amount of TNTs detected with CD34+ HSPCs (e.g., CD34+CD133high or CD34+CD133–/low cells) needs to be

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interpreted with caution. Our experiments with primary cells were performed in ex vivo cultures and the conditions might have been suboptimal in comparison with the native bone

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marrow niches. Second, TNTs are delicate structures and we cannot exclude that a fraction of them was lost during the recording, although the latter was performed with living, unfixed, cells. TNTs are rapidly destroyed upon the addition of paraformaldehyde (data not shown). Indeed, TNTs generated by KG1a cells might be more stable. We recently noted that primary cells from both acute myelogenous leukaemia and acute lymphoblastic leukaemia generated TNTs similarly to KG1a cells, suggesting that cancerous cells might share membrane properties distinct from that of non-transformed cells, e.g., CD34+ HSPCs (Reichert et al. unpublished data). Therefore, it will be interesting to evaluate the elastic and viscous

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properties of the plasma membrane underlying their deformability [76]. At first glance, the next step will be the determination of differences in cell stiffness between normal and transformed (cancerous) cells [77]. The levels of membrane cholesterol (or other lipids) that could influence the composition and/or organization of the plasma membrane, and hence its

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coupling to the submembrane cytoskeleton should also be determined in these cells [78]. What is the physiological consequence of the transfer of CD133 along TNTs? As mentioned in the Introduction the function of CD133 is currently unknown, the significance

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of its transfer between TNT-connected cells remains therefore a matter of speculation. Given that haematopoietic stem cell differenciation is associated with a reduction of CD133 [20], a

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phenomenon also observed with neural progenitors [79,80], the CD133 transfer might be the consequence of the differention process of migrating bone marrow-associated haematopoietic progenitor cells en route to the bloodstream. In contrast to the release of CD133+ membrane vesicles, which are taken up by MSCs [20], the transfer of CD133 between CD34+ progenitors through TNTs allows selectivity. Furthermore, the TNT-connected cell receiving

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CD133 could acquire new properties. The expression level of CD133 in distinct functional subsets of CD34+ progenitor cells (i.e. CD133high versus CD133–/low) could find substance in the context [31,32]. Knowing that CD133 highlights cells (normal and cancerous) with stem

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cell properties and that membrane rafts are involved in signal transduction [81,82], it is tempting to speculate that a specific CD133-containing membrane raft harbouring particular

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signalling pathways that are related to stem (progenitor) cells may be shared (or exchanged) between TNT-connected cells [83]. If such hypothesis turns out to be correct, it will obviously open a new area in the stem and cancer stem cell field research.

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ACCEPTED MANUSCRIPT Acknowledgments

We thank C.A. Fargeas for critically reading the manuscript. D.C. was supported by Deutsche Forschungsgemeinschaft (DFG, SFB655-B3; CO298/5-1) and Sächsisches Staatsministerium für Wissenschaft und Kunst (#4-7531.60/29/31). M.B. was supported by DFG (SFB655-B2).

and Genetics) for financial support.

Conflict of interest disclosure

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The authors declare no conflicts of interest.

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D.C. and D.R. are indebted to W.B. Huttner (Max-Planck-Institute of Molecular Cell Biology

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[55] Arndt K, Grinenko T, Mende N, et al. CD133 is a modifier of hematopoietic progenitor frequencies but is dispensable for the maintenance of mouse hematopoietic stem cells. Proc Natl Acad Sci USA. 2013;110:5582-5587. [56] Chauveau A, Aucher A, Eissmann P, Vivier E, Davis DM. Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells. Proc Natl

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Figure 1. Haematopoietic progenitor cells develop tunnelling nanotubes. (A-G) CD34+ HSPCs and KG1a cells were stained with membrane dye DiO (green) or DiI (red), mixed

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together as indicated and cultured on fibronectin-coated support for 2 hrs prior to live-cell confocal microscopy analysis (n > 4 and 7 for CD34+ HSPCs and KG1a cells, respectively). 3-dimensional reconstructions of 30-40 x-y sections (0.5-µm slice) and side projections (A-G)

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are shown. Dashed line indicates the coverslip level. Note that the entire TNTs between CD34+ HSPCs are exclusively generated from one cell (A, B, inset), whereas KG1a cells

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exhibit TNTs generated from either one (D) or two cells (E, inset). A very rare example of TNT produced by two CD34+ HSPCs is shown (C). TNT membranes in the contact zone seem nearby (yellow arrowhead) or more distant (green arrowhead) when CD34+ HSPCs and KG1a cells, respectively, were engaged. When co-cultured together, almost the entire TNT between CD34+ HSPCs and KG1a cells originated from latter cells and their ending points

cell. Scale bars, 10 µm.

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seemed to be distant (F, G, green arrowhead, n = 3). Asterisk marks the uropod of a migrating

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Figure 2. Morphological characteristics of tunnelling nanotubes. (A) CD34+ HSPCs were

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stained with membrane dye DiO (green) or DiI (red), mixed together and cultured on fibronectin-coated support for 2 hrs prior to live-cell confocal microscopy analysis. A 3dimensional reconstruction of 40 x-y sections (0.5-µm slice) and a side projection are shown. Dashed line indicates the coverslip level. Three red circles highlight small fluorescent bulges along a TNT. (B-D) CD34+ HSPCs (B) and KG1a cells (C, D) were cultured for 2 hrs on either MSCs (B, D) or fibronectin-coated support (C) prior the SEM analysis. Specific regions highlighted with coloured boxes in panels B-D are shown with a high magnification. Red circles indicate membrane bulges along TNTs. Dotted white lines and yellow arrowheads point out the apparent membrane continuity of TNT ending points with HSPC body (B),

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while dotted red line and green arrowheads show the junctional points of a TNT that links two KG1a cells (inset in C, D). Representative images from three independent preparations are shown. Scale bars, 10 µm; except inset (B, C), 100 nm.

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Figure 3. Tunnelling nanotubes are driven by F-Actin in a cholesterol-dependent manner. (AF) KG1a cells were stained, or not, with membrane dye DiI (red) or DiO (green), cultured on fibronectin-coated support and subjected to different treatments. Upon 2 hrs in culture, DiI-

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labelled cells were fixed and stained with phalloidin to visualize F-actin within TNTs (A, black arrowhead). Alternatively, unstained cells were incubated without (control) or with

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nocodazole (1 µM) for 2 hrs, and then fixed prior their labelling with phalloidin in combination with anti-α-tubulin antibody, which revealed the absence of microtubule (white arrowhead) in actin-positive TNT (B). DiO-/DiI-labelled cells were seeded and immediately incubated for 2 hrs with latrunculin B or nocodazole at various concentrations (C). The

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number of TNTs per 100 cells upon depolymerization of actin filaments or microtubules was counted using live-cell confocal microscopy analysis (≥80 cells were evaluated for each drug concentration used, n = 4 independent experiments). Additionally, cells were submitted to

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two treatment protocols (D, see schematic workflow) where the actin polymerization was inhibited (latrunculin B at 5 µM). The live-cell imaging analyses were performed after the

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treatment (imaging I and III), and upon the drug removal (imaging II). Optical sections at the bottom and middle of cells are shown; TNTs (black arrowheads) appear in the middle section while filopodium-like structures (grey arrowheads) at the coverslip level (D). TNTs are partly sensitive to cholesterol extraction (E, F). Cells were incubated without (control) or with MβCD (1 mM) for 10 up to 60 min, and TNTs and/or filopodium-like structures were observed using live-cell imaging (E, asterisks indicate cells without podia) and quantified (F, ≥40 cells were evaluated for each time point per experiment, n = 4). Statistical analyses were performed using two-tailed paired student’s t-test (**, p < 0.01; ***, p < 0.001). Scale bars, 10 µm.

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Figure 4. Influence of extracellular matrix-based substrata on the formation of tunnelling nanotubes. (A-D) CD34+ HSPCs and KG1a cells were stained with membrane dye DiO (green) or DiI (red) and cultured either on MSCs, fibronectin, collagen I or poly-L-lysine-

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coated support prior to live-cell confocal microscopy analysis. A 3-dimensional reconstruction of 30-40 x-y sections (0.5-µm slice) and a side projection (inset) of fluorescent images are shown (A-C). Asterisks and arrowheads indicate the uropod of a migrating cell

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and TNT, respectively. Time-lapse video microscopy revealed that cytonemes/magnupods are moving fast as illustrated by the lack of focus (D). The elapsed time is indicated in the upper

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right corner. Each substratum was analysed in quadruplet and the same data were observed. Alternatively, unlabelled cells were monitored by time-lapse video microscopy (A-C). Tracking diagrams of 50 individual cells for a 30 min period are depicted including their migration distances (in µm; mean ± standard deviation, bars = 20 µm, 15-20 cells per experiment, n = 3). In absence of cell migration, TNTs are rarely detected (B) in contrast to

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filopodia (green arrows) and cytonemes/magnupods (B, C, red arrows). Scale bars, 10 µm.

Figure 5. The biogenesis of tunnelling nanotubes relies on the membrane of uropod and cell

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migration. (A, B) DiO-/DiI-labelled or unlabelled KG1a cells cultured on fibronectin-coated

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support were observed either by fluorescence (left panel), SEM (middle panel) or bright-field (right panel) microscopy (A), or alternatively by time-lapse bright-field video microscopy (B). The elapsed time is shown in the upper right corner of each frame, and two migrating KG1a cells are pseudocoloured (B). Asterisks and yellow arrowheads indicate the uropod and TNTs, respectively. (C-E) Unlabelled (C, D) or DiO-/DiI-labelled (E) KG1a cells were treated with ROCK inhibitor Y-27632 (10 µM) or latrunculin B (5 µM) or without (control) for 2 hrs and analysed by time-lapse video microscopy for a 30 min period. Tracking diagrams of 50 individual KG1a cells for a 30 min period are depicted including their migration distances (in µm; mean ± standard deviation, bars = 20 µm) under various

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conditions (C, 15-20 cells per experiment, n = 3). Plots represent half of the data points within the box and minimum and maximum of all of the data within the whiskers (D). Horizontal lines within the box represent median values (50 cells, n = 3). The number of TNTs formed by 100 cells under these conditions was counted using live-cell confocal

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microscopy analysis (E, 100 cells, n = 4). Statistical analyses were performed using twotailed paired student’s t-test (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Scale bars,

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10 µm.

Figure 6. The characterization of CD34+ haematopoietic progenitor subpopulations reveals a

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connexion between tunnelling nanotube formation and cell morphology. (A) Schematic representation of the experimental setup to isolate HSPC subpopulations. CD133+ HSPCs from the leukapheresis product derived from single donors were enriched using CD133conjugated paramagnetic microbeads. The labelled CD133+ cells recovered from the first column were run over a second column (2x) in order to obtain a higher purity of cells

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harbouring CD34+CD133high (CD133-MACS). Cells in the first flow-through of the CD133 selection were collected and subjected to CD34 selection, leading to an enrichment of CD34+CD133– and CD34+CD133low cells (CD133–FT) as evaluated by flow cytometry. One

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representative experiment is shown. Median fluorescence intensity (MFI) for CD133 is

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indicated. (B-E) DiO-labelled cells incubated for 2 hrs on fibronectin coated support (B) or unlabelled (C-E) cells cultured on MSCs were observed either by live-cell confocal microscopy (B), time-lapse bright-field video microscopy (C, D) or SEM (E). The number of TNTs formed by DiO-labelled cells was counted for each HSPC fraction (B, 80000 cells per experiment, n = 3). Tracking diagrams of 20 individual cells are depicted including their migration distances (in µm; mean ± standard deviation, 50 cells were analysed per donor, n = 3, bars = 20 µm) from each HSPC subpopulation (C). Plot represents half of the data points within the box and minimum and maximum of all of the data within the whiskers. Horizontal lines within the box represent median values. Migrating cells with an elongated or

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rounded cell body are pseudocoloured in green and red, respectively (D, one frame of video is displayed) and quantified (235 cells for each cell fraction were evaluated per experiment, n = 3). Statistical analyses were performed using two-tailed unpaired student’s t-test (***, p < 0.001; ****, p < 0.0001). Cells with an elongated cell body (panels I and IV, green

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bracket) contain a uropod (green asterisk) at their backward (yellow dotted line), while those with a spherical morphology (panels II and III, red line) display a uropod-like structure (red asterisk) often at the apex of cell body (white dotted line) or are devoid of such structure

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(yellow star) (E). A lamellipodium is observed at the leading edge (LE) of all migrating cells. Note that some cells with rounded body are depicted in green (D, CD133-MACS) since they

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became elongated during migration and few elongated cells are found in CD133-FT fraction (D, E, panel IV). SEM images are representative data obtained from two independent donors. Scale bars, 25 (D), 10 (E, I, II), 1 (E, III, IV) µm.

Figure 7. Intracellular molecules are unable to pass the junctional complex of the TNT. KG1a

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cells or CD34+ HSPCs were stained with membrane (DiI, red) and cytoplasmic (CFSE, green) dyes, mixed together and cultured on fibronectin-coated support for 2 hrs prior to live-cell confocal microscopy analysis. 5-6 x-y sections (0.5-µm slice) taken at the middle level of

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cells are shown. The box displays the region of cell-cell contact at higher magnification. Note that neither membrane dye nor cytoplasmic labelled proteins crossed the junctional complex

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within the TNT (red dashed line). Asterisk marks the uropod of the cell. Representative images from two (CD34+ HSPCs from distinct donors) and four (KG1a cells) independent preparations are shown. Scale bars, 10 µm.

Figure 8. Solely CD133 displays clusters of signal along the tunnelling nanotubes. (A-C) KG1a cells were transfected with plasmids encoding either for GFP (green) or mCherry (red) protein fused to CD molecules or double transfected as indicated and cultured on fibronectincoated support prior their analysis by live-cell confocal microscopy. A single x-y section

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taken at the middle level of cells (A-C) and a side projection (A) or maximal projection of 3040 x-y sections (C) are shown. Differential interference contrast image (DIC) is added in the merge. Dashed line indicates the coverslip level. While the CD34, CD44, CD53 or CD63 fusion protein appeared as a continuous signal along the TNT (A, B, bracket, n = 3 (CD34,

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CD53, CD63) or 10 (CD44)), CD133-mCherry is clustered in discrete signals (A, white arrows, n = 18). CD133 is not found in all TNTs (B). In contrast to CD133-GFP (see below Fig. 8D), CD44-mCherry signal did not cross the junctional complex within the TNT (C, red

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dashed line and yellow box). A filopodium at the level of coverslip is indicated (C, yellow

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arrow). Scale bars, 10 µm.

Figure 9. Directional intercellular transfer of CD133. (A-E) KG1a cells were transfected with plasmids encoding for CD133-GFP (A-D) or other fusion proteins (E) and cultured on fibronectin-coated support prior to their analysis by live-cell confocal (A, B, E) or time-lapse video (C, D) microscopy. Typical examples of fluorescence x-y sections including their

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merge with differential interference contrast image (DIC) and their side projections are displayed (A, B) with an illustration (B). In panel A, a TNT is enlarged (cyan box). Cell borders and the coverslip are indicated with dotted and dashed lines, respectively. Punctate

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GFP signals (white arrows) are observable along TNTs between a CD133-GFP–expressing

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cell (I) and the TNT-connected one (II). A faint and continuous labelling is also observed (brackets). Discrete CD133-GFP signals are detected in cell II (A, B, red arrows) or at the tip of a membrane protrusion in contact with the substrate (B, yellow arrow). Time-lapse video microscopy analyses reveal the directional transfer (cyan arrow) of CD133-GFP between cells I and II (C, D). The elapsed time is shown in the lower left corner. To facilitate the analysis, fluorescent images are reproduced in black with a white background. The initial and final positions of cells are highlighted with yellow and black/white dotted lines. CD133-GFP signals transferred to cell II are indicated with red arrows. In some cases, an accumulation of CD133-GFP signal was observed at a specific and stationary point within the TNT (D, red

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dashed line, STOP) with the directional movement of punctate CD133-GFP occurring before and after it (orange and green arrows, respectively). For comparison, DiO-/DiI-labelled cells are displayed in the right panel (D). Cells transfected with miscellaneous GFP-fusion proteins show a continuous GFP signal along the TNT and no signal is observed in TNT-connected

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cell (E, dotted lines). Representative images or videos from four independent preparations are shown. Scale bars, 10 µm.

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Figure 10. Transfer of CD133 occurs via the cell surface of tunnelling nanotubes. (A, B) CD133-GFP transfected KG1a cells (A) or WGA-FITC labelled CD34+ HSPCs (B) were cell

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surface labelled with anti-CD133 APC-coupled antibody (αCD133-APC) and after 15 min analysed by live-cell confocal microscopy. 40 x-y sections including their merge with differential interference contrast image (DIC, A, B), and a side projection below the corresponding cartoon (A) are shown. Specific regions highlighted with coloured boxes are

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displayed at higher magnification. Cell borders and the coverslip level are indicated with dotted and dashed lines, respectively. Note that the punctate CD133-GFP signal (arrows) along a TNT and a fraction of them (yellow arrowhead) associated with the cell body are detected with αCD133-APC indicating their presence at the cell surface (A). In contrast, a

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discrete and more continuous CD133-GFP signal is not labelled with αCD133-APC

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(bracket). The cell surface-associated punctate CD133 staining is observed in TNT between connected CD34+ HSPCs (B, white arrowheads). (C) DiO-labelled KG1a cells were fixed, permeabilized and stained with phospho-myosin light chain (MLC) 2 antibody and DAPI prior to confocal microscopy analysis. A TNT is displayed at higher magnification in the cyan box. Like CD133, phospho-MLC2 appears as a punctate staining (white arrowheads). Asterisk mark the uropod. Scale bars, 10 µm.

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Figure 11. A potential mechanism underlying the selective transport of CD133 along tunnelling nanotubes. The biological properties of the five-transmembrane (1-5) glycoprotein CD133 might modulate the lipid composition and the local organization of the plasma membrane within TNTs. For instance, i) its direct interaction with membrane cholesterol; ii)

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its incorporation into cholesterol-rich membrane rafts; iii) its preference for membrane protrusions with a strong curvature, and iv) its Src/Fyn-mediated phosphorylation (P) at the conserved C-terminal cytoplasmic residue tyrosine, which promotes its interaction with the

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PI3K 85 kDa regulatory subunit resulting in the activation of PI3K, could explain its directional transport (green arrow) along TNTs. The PI3K activation would lead to the

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conversion of docking phospholipids PIP2 to PIP3 and the putative interaction of PIP3enriched membrane cholesterol–CD133 complexes with actin motor protein myosin might promote their selective movement along the actin filament. The CD133–containing micro/nanodomain (red) is drawn thicker than the surrounding membrane [84], and for simplicity, the cholesterol is not depicted. The nature of the putative interaction of myosin

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molecules with membrane rafts and the mechanism that could explain their transfer through

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ACCEPTED MANUSCRIPT Highlights
Haematopoietic progenitors are interconnected by F-actin-dependent tunnelling nanotubes
Cell dislodgement is the primary mechanism responsible for tunnelling nanotube biogenesis
Tunnelling nanotubes emerge from the uropod of migrating ha matopoietic progenitors

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The stem cell marker CD133 is transferred between ha matopoietic cells via tunnelling nanotubes

ACCEPTED MANUSCRIPT SUPPLEMENTARY DATA Tunnelling nanotubes mediate the transfer of stem cell marker CD133 between haematopoietic progenitor cells Doreen Reichert, Julia Scheinpflug, Jana Karbanová, Daniel Freund, Martin Bornhäuser

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and Denis Corbeil

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SUPPLEMENTARY FIGURES

Supplementary Figure S1. The addition of SDF-1α (CXCL12) did not increase the number of tunnelling nanotubes. (A) Schematic workflow of protocols used to evaluate the effect of SDF-1α

cell migration and TNT formation. Prior to live-cell imaging analyses, cells

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ACCEPTED MANUSCRIPT were incubated without (control) or with SDF-1α (100 ng/ml) either immediately after seeding (imaging I) or 2 hrs later (imaging II). (B-G) Unlabelled (B, C, E, F) or DiO-labelled (D, G) KG1a cells (B-D) or CD34+ HSPCs (E-G) cultured on fibronectin were incubated according to the protocol described in panel A. Unlabelled cells were monitored by time-lapse

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video microscopy. Tracking diagrams of 50 representative individual cells for a 30 min period are depicted (B, E) including their migration distances (in µm; mean ± standard deviation, n = 100-120 cells, bars = 20 µm). Plots represent half of the data points within the box and minimum and maximum of all of the data within the whiskers. Horizontal lines within the box

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represent median values (C, F). The number of TNTs formed by DiO-labelled cells under

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these conditions was counted using live-cell confocal microscopy analysis (D, 200 cells; G, 80000 cells). Statistical analyses were performed using two-tailed paired student’s t-test (***, p < 0.001; n.s., not significant).

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Supplementary Figure S2

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Supplementary Figure S2. Breakage of tunnelling nanotubes as an alternative mechanism of transfer of materials. KG1a cells were stained with membrane (DiI, red) and cytoplasmic (CFSE, green) dyes, mixed together and cultured on fibronectin-coated support for 2 hrs prior live-cell confocal microscopy analysis. Single (I) and composite of 30-40 x-y sections (II, left panel) with side projections (II, right panel, III) are shown. Note that DiI-labelled membrane fragments (arrowhead) are found in the opposite CFSE-labelled cells. White dashed line indicates the coverslip level. Scale bars, 10 µm.

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Supplementary Video S1. Video analysis showing that the cell dislodgment mechanism is responsible for biogenesis of tunnelling nanotubes. KG1a cells were cultured on fibronectin-

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coated support prior to recording by time-lapse bright-field video microscopy. The elapsed time is shown in the upper left corner. Total video time: 21 min. Stills from this movie are shown in Fig 5B. (Format: mov; size: 3.3 MB)

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Supplementary Video S2. Video analysis showing CD34+CD133+ cells (CD133-MACS) turning around the contact point between the uropod and the feeder cell layer. CD34+

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subpopulation harbouring CD133 was isolated by MACS using CD133 antigen and cultured on MSCs prior to recording by time-lapse bright-field video microscopy. The video depicts two elongated HSPCs that stopped migrating and started to rotate upon contact of their backward structure (uropod) with the MSCs. The elapsed time is indicated in the upper left

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corner. Total video time: 30 min. (Format: mov; size: 375 KB)

Supplementary Video S3. Video analysis showing the intercellular transfer of CD133-GFP.

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CD133-GFP transfected KG1a cells were cultured on fibronectin-coated support prior to recording by time-lapse video microscopy. The merged view of the phase contrast and

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fluorescence images is presented. Black and red arrows indicate CD133-GFP proteins along the TNT and the receiving cells, respectively. Total video time: 61 sec. Stills from this movie are shown in Fig 9C. (Format: mov; size: 507 KB)

Supplementary Video S4. Video analysis showing the stationary CD133-GFP+ bulge observed along the tunnelling nanotube. CD133-GFP transfected KG1a cells were cultured on fibronectin-coated support prior recording by time-lapse video microscopy. The view of fluorescence images is presented. Orange and green arrows indicate CD133-GFP proteins before and after the stationary CD133-GFP+ bulge observed along the TNT, respectively.

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