GRP75 upregulates clathrin-independent endocytosis through actin cytoskeleton reorganization mediated by the concurrent activation of Cdc42 and RhoA

Experimental Cell Research 343 (2016) 223–236

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Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr

Research article

GRP75 upregulates clathrin-independent endocytosis through actin cytoskeleton reorganization mediated by the concurrent activation of Cdc42 and RhoA Hang Chen a, Zhihui Gao a, Changzheng He a, Rong Xiang b, Toin H. van Kuppevelt c, Mattias Belting d, Sihe Zhang a,b,n a

Department of Biochemistry & Cell Biology, School of Medicine, Nankai University, Tianjin, PR China State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, PR China Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands d Department of Clinical Sciences, Section of Oncology, Lund University, Lund, Sweden b c

art ic l e i nf o

a b s t r a c t

Article history: Received 29 November 2015 Received in revised form 5 April 2016 Accepted 12 April 2016 Available online 22 April 2016

Therapeutic macromolecules are internalized into the cell by either clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). Although some chaperone proteins play an essential role in CME (e.g. Hsc70 in clathrin uncoating), relatively few of these proteins are functionally involved in CIE. We previously revealed a role for the mitochondrial chaperone protein GRP75 in heparan sulfate proteoglycan (HSPG)-mediated, membrane raft-associated macromolecule endocytosis. However, the mechanism underlying this process remains unclear. In this study, using a mitochondrial signal peptidedirected protein trafficking expression strategy, we demonstrate that wild-type GRP75 expression enhanced the uptakes of HSPG and CIE marker cholera toxin B subunit but impaired the uptake of CME marker transferrin. The endocytosis regulation function of GRP75 is largely mediated by its subcellular location in mitochondria and is essentially determined by its ATPase domain. Interestingly, the mitochondrial expression of GRP75 or its ATPase domain significantly stimulates increases in both RhoA and Cdc42 activation, remarkably induces stress fibers and enhances filopodia formation, which collectively results in the promotion of CIE, but the inhibition of CME. Furthermore, silencing of Cdc42 or RhoA impaired the ability of GRP75 overexpression to increase CIE. Therefore, these results suggest that endocytosis vesicle enrichment of GRP75 by mitochondria trafficking upregulates CIE through an actin cytoskeleton reorganization mechanism mediated by the concurrent activation of Cdc42 and RhoA. This finding provides novel insight into organelle-derived chaperone signaling and the regulation of different endocytosis pathways in cells. & 2016 Elsevier Inc. All rights reserved.

Keywords: GRP75 Clathrin-independent endocytosis ATPase domain Cdc42 RhoA Actin cytoskeleton

1. Introduction Endocytosis is an essential cellular process that is used by cells to internalize a diverse array of cargo molecules. Numerous cellular processes, including those of microbial invasion, nutrient

Abbreviations: CME, Clathrin-mediated endocytosis; CIE, Clathrin-independent endocytosis; HSPG, Heparan sulfate proteoglycan; ΔS, Without mitochondrialtargeting signal peptide; ATPaseD, ATPase domain; SBD, Substrate-binding domain; MAC, Membrane attack complex; GDI, Guanine nucleotide dissociation inhibitor; GPI-APs, GPI-anchored proteins; Ab, Antibody; αHS, Anti-heparin sulfate (HS) single chain antibody fragment; Tfn, Transferrin; CTxB, Cholera toxin B subunit; RBD, Rho-binding domain n Corresponding author at: Department of Biochemistry & Cell Biology, School of Medicine, Nankai University, 94 Weijin Road, Nankai District, Tianjin 300071, PR China. E-mail address: [email protected] (S. Zhang). http://dx.doi.org/10.1016/j.yexcr.2016.04.009 0014-4827/& 2016 Elsevier Inc. All rights reserved.

uptake, membrane receptor trafficking, cell motility and macromolecular drug delivery, are modulated by endocytosis through several distinct mechanisms. Although the exact definition of these mechanisms is still debated, a specified cargo molecule can be taken up by mammalian cells through clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). In comparison with the well-defined CME pathway, a more comprehensive understanding of CIE pathways based on their associations with/dependence on dynamin, caveolin-1, flotillin, Arf6 or the Rac1, RhoA, and Cdc42 Rho GTPase family has been achieved in recent years [1]. Interestingly, almost all CIE pathways appear to be triggered by and dependent on the actin cytoskeleton reorganization machinery because endocytosis through such pathways is sensitive to agents that inhibit actin filament polymerization. The housekeeping function of chaperone proteins has long

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been recognized to prevent aberrant protein aggregation by facilitating the assembly/disassembly and folding/refolding of cytoskeletal proteins [2]. Nonetheless, some chaperone proteins, particularly member of the Hsp70 family, not only facilitate cytoskeletal structure remodeling in abnormal situations, but also play specific roles under normal conditions to regulate a variety of cell endocytosis activities. For instance, the cytoplasmic Hsp70 protein (Hsc70/HspA8) is widely known to drive multiple phases of CME and in particular catalyzes the disassembly of clathrin cages together with co-chaperones like auxilin (DnaJC6) or GAK (Cyclin G-associated kinase/DnaJC28) [3]. The endoplasmic reticulum Hsp70 protein (BiP/Grp78/HspA5) has frequently been reported to function as a cell surface-binding receptor or co-receptor for the endocytosis of several viruses and matrix proteins, a process that is always mediated by caveolae or lipid rafts [4–6]. The mitochondrial Hsp70 protein (Grp75/Mortalin/HspA9) has been shown to bind directly or associate with certain cytokine or cytokine receptors thereby mediating their intracellular trafficking or endocytosis [7,8], typically via RhoA-dependent CIE routes. Thus, it appears that Hsp70 chaperone proteins play an active role in different endocytosis pathways depending on their specific intracellular localizations. Various macromolecules, viruses and ligand complexes enter cells via cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis, which involves the use of a glycosyl-phosphatidyl-inositol (GPI)-linked glypican (GPC) or transmembrane syndecan (SDC) as the initial docking receptor. We previously provided the first demonstration that mitochondrial chaperone protein GRP75 is enriched as a functional constituent in HSPGmediated and membrane raft-associated endocytosis vesicles. Notably, the role of GRP75 in this clathrin-independent but Rho GTPase Cdc42-dependent endocytosis pathway has been revealed through the use of RNAi-mediated knock-down method [9]. GRP75, a multifunctional heat shock protein 70, plays an essential role in the importing, sorting and refolding of mitochondrial matrix proteins [10]. Its trans-localization to outside the mitochondria results in its collaboration with a repertoire of binding partners while playing an expansive cellular role. For example, cytosolic GRP75 has been isolated as a protein that binds to endocytic FGF-1 [7], associates with IL-1 receptor type I (IL-1R1) [8], and colocalizes with mannose receptor (MR) [11]. All of these interactions have been believed to facilitate intracellular trafficking and/ or signaling cascades. Moreover, cell surfaces associated with GRP75 have been found to bind components of C5b-9 complexes while promoting the shedding or uptake of membrane vesicles loaded with complement MAC (membrane attack complex). Interestingly, the cytosolic overexpression of ΔS GRP75 (lacking the 51 N‐terminal amino acids corresponding to the mitochondrial targeting sequence) exhibits limited protective capacities against CDC (complement-dependent cytotoxicity) relative to that of mitochondrial GRP75. Furthermore, although GRP75 interactions with complement proteins C8 and C9 have been determined based on the ATPase domain (ATPaseD), this is not its substrate-binding domain (SBD) [12]. For the HSPG-mediated and membrane raftassociated uptake of macromolecules, is the regulation function of GRP75 in endocytosis affected by its subcellular location? Which domain of the GRP75 protein determines its regulatory function? What is the specific relationship between GRP75 and Cdc42 activation in this pathway? Are other Rho GTPases also involved? Is the actin cytoskeletal organization affected by the subcellular distribution of GRP75? All of these questions remain to be clarified. In this study, using a signal peptide-directed protein ectopic expression approach, we found that the mitochondrial expression of GRP75, mainly through its ATPaseD, significantly promoted CIE while simultaneously inhibiting CME. The expression of GRP75 or

ATPaseD in mitochondria induces the concurrent downstream activation of RhoA and Cdc42, and thereby enhances the formation of stress fibers and filopodia, effects that contributed collectively to the promotion of CIE and the inhibition of CME. Together, our results provide the first demonstration that GRP75 subcellular translocation/trafficking/enrichment upregulates CIE through an actin cytoskeleton reorganization mechanism mediated by the concurrent activation of Cdc42 and RhoA.

2. Materials and methods 2.1. Plasmids, siRNAs, antibodies and reagents Human wild type (wt) GRP75-pEGFP plasmid [13] was kindly provided by Prof. Kenji Fukasawa (H. Lee Moffitt Cancer Center & Research Institute). pCMV5-FLAG-Cdc42 (WT), pCMV5-FLAGCdc42 (G12V) and pCMV5-FLAG-Cdc42 (T17N) plasmids [14] were kindly provided by Prof. Takaya Satoh (Osaka Prefecture University). pGEX-2T-GST-Rhotekin RBD and pGEX-GST-PAKCRIB RBD plasmids [15] were kindly provided by Prof. Alan Hall (Memorial Sloan-Kettering Cancer Center). QIAquick Gel Extraction Kits and QIAquick PCR Purification Kits were obtained from Qiagen Ltd. The restriction enzymes BspE I and Hind III, T4 DNA ligase and Phusions high-fidelity DNA polymerase were obtained from New England Biolabs Ltd. GRP75, Cdc42 and RhoA siRNA oligo pools (a mixture of three siRNA duplexes. Table S1) were obtained from GenePharma Ltd. The primary Abs used included mouse antiGRP75 (SC-133137, Santa Cruz), rabbit anti-GRP75 (SAB4501454, Sigma), goat anti-GRP75 (SC-1058, Santa Cruz), rabbit anti-Cdc42 (SC-87, Santa Cruz), mouse anti-RhoA (SC-418, Santa Cruz), rabbit anti-RhoA (SC-179, Santa Cruz), rabbit anti-Rac1 (SC-217, Santa Cruz), rabbit anti-b-actin (4970S, Cell Signaling), rabbit anti-Flag (F742S, Sigma), goat anti-Flag (ab1257, Abcam), mouse anti-FLAG (M185, MBL) mouse anti-VSV Ab P5D4 (V5507, Sigma), mouse-GFP (sc-9996, Santa Cruz), rabbit anti-GFP (2956, Cell Signaling), and mouse anti-GST (SC-138, Santa Cruz). A single chain variable fragment Ab AO4B08 (scFv-αHS) was obtained by biopanning against heparin sulfate (HS) isolated from mouse skeletal muscle cells [16]. Other reagents used included mitochondria-selective probe MitoTrackers Red CMXRos (M7512), Tfn-AF647 (T-23366), CTxB-AF647 (C-34778), goat anti-mouse Ab-Alexa Fluor 488 (A-11001), goat antirabbit Ab-Alexa Fluor 488 (A-11070), rabbit anti-goat Ab-Alexa Fluor 488 (A-11078), rabbit anti-goat Ab-Alexa Fluor 546 (A-21085), goat anti-rabbit Ab-Alexa Fluor 568 (A-11031), Lipofectamines 2000 (11668027), Lipofectamines RNAiMAX (13778075), Dynabeadss Protein G (10003D) and BCA Protein Assay Kits (23225), all of which were obtained from Life Technologies. Glutathione Sepharose™ 4B (17075601) was obtained from GE. An EDTA-free Protease Inhibitor Complete Mini (04693124001) was obtained from Roche. Rhodamine-phalloidin (PHDR1) was obtained from Cytoskeleton. Latrunculin A (Lat A, L5163), poly-D-lysine (P7886), cell media, supplements and the remaining fine-grade chemicals used in this study were obtained from Sigma-Aldrich. 2.2. Functional domain constructs Full-length GRP75 gene fragments without mitochondria-targeting signal peptides (ΔS GRP75), ATPase domains with/without signal peptide (ATPaseD/ΔS ATPaseD), and SBD domains without signal peptides (ΔS SBD) were PCR-amplified from pEGFP-GRP75 (wt) plasmid using the following respective primer pairs: 51BF (5′-CAGCCATCCGGAATCAAGGGAGCAGTTGTTGG-3′) and 679R (5′-ATTATAAGCTTCTGTTTTTCCTC- CTTTTGATCTTCC-3′), 1SF (5′-CAGCCATCCGGAATGATAAGTGCCAGCCGAGCTG-3′) and 436R (5′-AT-

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Fig. 1. Identification of GRP75 constructs with/without mitochondrial-targeting signal peptides. (A) Schematic representation of GRP75 and its limited-domain constructs. wt: wild type. SP: mitochondrial-targeting signal peptide. ΔS: without signal peptide. ATPaseD: ATPase domain. SBD: substrate-binding domain. (B) HeLa cells were transiently transfected with the EGFP recombinant plasmids listed above. Forty-eight hours after transfection, the cell lysates were analyzed through SDS-PAGE followed by western blotting with rabbit anti-GFP Ab (1:2000) and mouse anti-GRP75 Ab (1:1000) respectively. (C) The HeLa cells transfected with the listed plasmids were first stained with MitoTrcker (300 nM) for 10 min at 37 °C for mitochondria labeling (red color). After fixation and permeabilization, untransfected cells (leftmost image in up row) were sequentially stained with mouse anti-GRP75 (1:100) and then with anti-mouse AF488 (1:1000, green color); Plasmids-transfected cells were sequentially stained with rabbit anti-GFP Ab (1:1000) and then with anti-rabbit AF488 (1:1000). Finally, all co-stained cells were examined under a confocal microscope. Representative cells showing the colocalization of either endogenous GRP75 or exogenous EGFP-fused proteins with mitochondria are shown (yellow color) for each transfection. Scale bar, 20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

TATAAGCTTCAGCACATCCGTGACATCGC-3′), 51BF and 436R, 434BF (5′CAGCCATCCGGAGATGTGC- TGCTCCTTG-3′) and 679R. To generate the SBD domain, a signal peptide sequence was amplified with the 1SF and 50C2R (5-CCAACAACTGCTCCCTTGATTGCTTCTGATGCATAATCCCG-3′) primer pairs and was fused with a ΔS SBD fragment via overlap extension PCR using high-fidelity DNA polymerase. The PCR products were fractionated using 1.5% agarose gel, isolated using Qiaquick@ Gel purification kit, double-digested with BspE IþHind III, purified using QIAquick PCR purification kit, and were finally cloned

into the pEGFP plasmid using T4 DNA ligase. All recombinant plasmids (Fig. 1A) were checked via double digestion with BspE IþHind III, and the ORF was determined through DNA sequencing (Sangon Ltd.). 2.3. Cell culture and treatments HeLa and NIH3T3 cells from ATCC were cultured in DMEM medium containing 10% (v/v) FBS and maintained at 37 °C with 5%

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(v/v) CO2. The cells were transient-transfected with EGFP-fused plasmids (pEGFP as control) and/or transfected with GRP75/ Cdc42/RhoA specific siRNA oligo pool (scrambled siRNA as control) for 6 h using Lipofectamines 2000 or RNAiMAX, then reseeded and were cultured for 48–72 h before analysis. For the flow cytometry and confocal fluorescence assays, 0.3 μg of plasmid DNA was added to each well in a 48-well plate or chamber slide, unless otherwise stated. For the co-immunoprecipitation and Cdc42/ RhoA/Rac1 activation assays, 5 μg of plasmid DNA was used for each 100 mm dish. For the RNA interference (RNAi) assay, 100 nM (final concentration) siRNA pool was used in each transfection. Both the overexpression and knock-down efficiency were assessed by western blotting. For the actin polymerization inhibition assay, the dose of actin perturbants was carefully titrated to fulfill significant inhibition of CIE marker Cholera toxin B subunit (CTxB) but not on CME marker transferrin (Tfn). Cells were treated with Lat A (5 μM) or carrier (dimethyl sulphoxide) for a period of 5 min at 37 °C prior to the analysis of endocytic capacity. 2.4. Western blotting and immunoprecipitation Cells were washed with cold PBS and then lysed in standard lysis buffer (50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS). Cell debris was removed via centrifugation at 13,000 rpm for 10 min and protein concentration of the lysate was measured using the BCA assay. Protein samples were resolved by SDS-PAGE, blotted onto PVDF membranes and then analyzed with different Abs as noted in the text. For the immunoprecipitation experiments, the cells were washed with cold PBS and then lysed in IP lysis buffer (10 mM Tris, pH 7.4, 140 mM NaCl, 5 mM EDTA, 25 mM NaF, 10 mM sodium pyrophosphate, and 1% NP-40 freshly supplemented with 1 mM sodium orthovanadate, 5 mM sodium glycerophosphate and one complete tablet/50 ml). The cell lysate (200 μg total proteins) were precleared for 1 h with Dynabeadss protein G, incubated with 2 μg of Ab overnight at 4 °C, and then incubated with Dynabeadss protein G for an additional 1 h of incubation. The beads were washed four times with IP lysis buffer before being loaded onto gels. 2.5. Flow cytometry The transfected cells were serum-free starved for 30 min, then live cells based flow cytometry was used to check the binding and uptake of αHS (1:20), CTxB (10 mg/ml) and Tfn (25 mg/ml). For the binding experiments, the cells were first detached using PBS (2  )/ 0.5 mM EDTA, washed twice with PBS BSA (1% w/v) and incubated with αHS, then washed in PBS BSA and incubated with mouse anti-VSV Ab (1:500), followed by rinsing in PBS BSA and incubation with goat anti-mouse-AF647 Ab (1:200). All binding incubations with three ligands were performed for 30 min on ice. Finally, cells were washed in PBS BSA and analyzed immediately on FACSCalibur instrument integrated with Cell-Quest software. For the uptake experiments, αHS, mouse anti-VSV (1:500) and goat anti-mouse-AF647 Ab were pre-complexed in serum-free medium at 20 °C for 30 min (αHS-AF647 complex), then incubated with cells at 37 °C for 1 h (30 min incubation for CTxB-AF647, and 15 min incubation for Tfn-AF647). Cells were then trypsinized, suspended in cold DMEM 10% FBS, washed with cold PBS BSA and analyzed immediately by flow cytometry. Controls without αHS primary Ab were included in all experiments. In some experiments, the cells were co-transfected with EGFP recombinant plasmids and siRNA pools, then cultured for another 48 h for reversing the overexpression of GRP75. The binding and uptake ofαHS-AF647 complex, CTxB-AF647 and Tfn-AF647 were determined by live cells-based flow cytometry through gating of EGFP expressing and –nonexpressing (or –low expressing) cells. In

the case of combined treatments with Cdc42 dominant-active overexpression or Cdc42 RNAi followed by GRP75 overexpression or its RNAi, the uptakes were compared in transfected cells only with mono-treatment. 2.6. Confocal microscopy and image analysis The transfected cells were re-seeded in chamber slides, serumfree starved for 30 min, and then incubated with αHS-AF647 complex, CTxB-AF647 and Trfn-AF647 for 10–30 min at 37 °C. The cells were then rinsed three times with PBS supplemented with 1 M NaCl to remove surface-associated ligands and fixed in 3.7% (w/v) paraformaldehyde for 10 min A DAPI (1:1000) nuclear stain was performed after cell-permeabilization with 0.1% Triton X-100. The cells were mounted, observed and images were acquired using Olympus FV 1000 laser scanning confocal microscope equipped with 63  /1.4N.A. and 100  /1.44N.A. oil immersion lens. Sixteenbit z-series of confocal sections (step size ¼0.42 mm for 63  objective and 0.29 mm for 100  objective) were acquired in the photon-counting mode, and these acquisition parameters were kept identical across all samples. For image presentation, eight-bit maximal projections of the z-series were created using ImageJ software, and brightness was adjusted across the entire image using Volocity software. For quantification of the uptakes of αHS-AF647 complex, CTxBAF647 and Trfn-AF647 in cells, raw 16-bit z-series were first converted to sum slice projections, which represent the total sum value of the integrated fluorescence intensities of the entire z-series, using ImageJ. Individual cells were next outlined as regions of interest (ROIs), and the total integrated fluorescence intensities (Icell) were measured using Volocity software. The mean intensity of an ROI outside of the cell was also measured to serve as background intensity (Iback), and the relative fluorescence intensity (Irel) was calculated (Irel ¼[Icell–Iback/Aback*Acell]/Acell, where A represents the pixel area of the ROI). Resultant data was normalized against the average values of control samples. It was empirically determined that the cells with the 15% highest and lowest fluorescence intensities reliably represented mechanically broken or dead cells, respectively, so these cells were excluded from the analyses. For each experiment, at least 30 cells in total were analyzed. For staining of mitochondria and EGFP fused protein expression, the transfected cells were incubated first with MitoTrackers Red CMXRos (300 nM) in a complete medium at 37 °C for 10 min, then fixed with 3.7% (w/v) paraformaldehyde and permeabilized with 0.1% Triton X-100 followed by sequential staining with rabbit anti-GFP Ab and anti-rabbit-Alexa Fluor 488 Ab. To stain the actin cytoskeleton, transfected cells were re-seeded in poly-D-lysine (100 mg/ml) pre-coated chamber slides, serum-free starved, then fixed in 3.7% (w/v) paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100. Polymerized actin was stained with Rhodamine-conjugated phalloidin (100 nM) for 45 min at room temperature. Isotype-matched control IgG was included in all experiments. 2.7. Cdc42/RhoA/Rac1 activation assay Assays were created following the GST-RBD (Rho-binding domains) pull-down method [17]. In brief, GST-TRBD (Rhotekin RBD) and GST-PAK CRIB proteins were produced by expression in E. coli BL21 DE3plys and BL21 cells, respectively. GST fusion proteins were purified and loaded on Glutathione Sepharose™ 4B according to the manufacturer's instruction. Serum-starved cells growing on a six-well plate were scraped with ice-cold Lysis buffer B (for Rac1 and Cdc42 activation) or Lysis buffer C (for RhoA activation) and centrifuged for the removal of cell debris. One milliliter of

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beads pre-cleared cell lysate was then incubated with 20 mg of GST-PAK-CRIB beads (for Cdc42/Rac activation) or GST-RhotekinRBD beads (for RhoA activation) for 60 min at 4 °C. The beads were washed four times with Tris wash buffer B. The beads pellets were boiled for 5 min in 50 ml of SDS-PAGE sample buffer. Samples were run in parallel with total cell lysate and immunoblotted with Abs for the appropriate small GTPase (rabbit anti-Cdc42 Ab, 1:250; mouse anti-RhoA Ab, 1:250; rabbit anti-Rac1 Ab, 1:200) using the ECL detection method. The activation of Cdc42, RhoA and Rac1 (GTP-bound state) in the control and transfected cells was determined by densitometry, and total lysates were used for normalization of the total quantity of small GTPase protein. 2.8. Statistical analyses Microscopy, flow cytometry and western blotting data were derived from at least three independent experiments. All of the data obtained from the experiments were analyzed and are presented as the mean 7 SEM. For two-sample comparisons against the controls, unpaired Student's t-tests were used unless otherwise noted. One-way analysis of variance with a Dunnett's multiple comparison was used to evaluate the statistical significance of at least three groups of samples. Graphs were created using GraphPad Prism 5 software.

3. Results 3.1. Expression of GRP75 and its limited-domain plasmids with/ without signal peptides Endogenous GRP75 proteins, which include mitochondrialtargeting signal peptides within their precursors, are generally localized in mitochondria. However, recent advances reveal that GRP75 serve distinct function from the related heat shock proteins (HSPs), and it can be actively translocated to other cellular locations and assume novel functions controlling signaling, proliferation, invasion, apoptosis, inflammation and immunity [9,10,18]. To mimic the subcellular translocation of GRP75 protein and to further explore whether differences in its distribution affect the endocytosis of macromolecules, we first constructed GRP75 and its limited-domain plasmids with/without signal peptides (Figs. 1A and S1) and then examined their subcellular locations in HeLa cells following expression. Western blot analyses showed that all EGFP recombinant plasmids were expressed with the correct-size products based on the predicted molecular weights in cell lysates (Fig. 1B). No significant differences in fusion protein expression levels were detected between the mitochondrial signal peptides containing plasmids and the corresponding mitochondrial signal peptides lacking plasmids (wtGRP75 vs ΔSGRP75, ATPaseD vs ΔSATPaseD, and SBD vs ΔSSBD) (Fig. 1B). To determine whether the EGFP fusion GRP75 or its limited-domain plasmids were expressed in the mitochondria, all transfected HeLa cells were labeled using MitoTracker Red. Confocal microscope analyses revealed substantial quantities of expressed wtGRP75 in mitochondria (Fig. 1C). In contrast, the absence of a mitochondria-targeting sequence in ΔSGRP75 led to its protein expression evenly distributed throughout the cytoplasm rather than in mitochondria (Fig. 1C). This considerable difference in subcellular distribution patterns was also found for the expression of limited-domain construct pairs (ATPaseD vs ΔSATPaseD, SBD vs ΔSSBD; Fig. 1C). These results indicate that mitochondria-targeting signal peptides essentially direct the enrichment/trafficking of expressed GRP75 or its limited-domains and determine their final subcellular locations in mitochondria.

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3.2. GRP75 promotes clathrin-independent endocytosis but inhibits clathrin-mediated endocytosis To explore the functional role of GRP75 in endocytosis pathway, HeLa cells were transfected with increasing concentrations of wtGRP75 plasmid and were then treated with a GRP75 siRNA pool (Fig. 2A). After the incubations with αHS-AF647 complex, CME marker Tfn-AF647 and membrane raft marker CTxB-AF647, flow cytometry and confocal microscope analyses of the EGFP-positive cells showed that wtGRP75 expression increased the uptake of αHS-AF647 complex (average increments of 10–110% and 10–140% were obtained in the FCM and confocal analyses, respectively) and CTxB-AF647 (average increments of 15–135% and 40–160% were obtained in the FCM and confocal analyses, respectively) but limited the uptake of Tfn-AF647 (average decrements 8–45% and 10– 40% were obtained in the FCM and confocal analyses, respectively) in a concentration-dependent manner (Figs. 2C and S2B). Notably, the expression-concentration-dependent uptake of αHS-AF647 complex and CTxB-AF647 was completely reversed by GRP75targeting RNAi (Figs. 2C and S2B). No significant changes in the αHS-AF647 complex, Tfn-AF647 and CTxB-AF647 binding properties were observed after the expression or knock-down of wtGRP75 (Figs. 2D, E, F and S2C). These results show that GRP75 expression promotes the uptake of αHS and CTxB but reduces the uptake of Tfn, complementing our previous observations [9] and strongly suggesting that GRP75 conversely regulates CIE and CME. 3.3. GRP75 promotes clathrin-independent endocytosis through its ATPase domain Similar to other proteins in the Hsp70 chaperone family, GRP75 has two functional domains, an N-terminal ATPaseD and a C-terminal SBD, which are joined by a protease-sensitive site [10]. In principle, ATPaseDs of Hsp70 proteins are preserved, and exertion of their chaperone activity requires ATP hydrolysis, whereas SBDs show more significant sequence variations, leading to diversification of client proteins and substrate specificities. To examine which domain of GRP75, i.e., the ATPaseD or SBD, determines its regulation activity, HeLa cells were transfected with wtGRP75, ATPaseD and SBD plasmids, and the uptake patterns of αHS-AF647 complex, Tfn-AF647 and CTxB-AF647 were then compared. Compared with wtGRP75, ATPaseD expression alone showed a similar capacity to increase the uptake of αHS-AF647 and CTxB-AF647 (Figs. 2D, E, 3B, C, S3A and S3B) but inhibited TfnAF647 uptake (Figs. 2F, 3D and S3C). In contrast, the patterns obtained by SBD transfection revealed a discrepancy between the FCM and confocal results: SBD expression only resulted in αHSAF647 uptake inhibition (Figs. 2D, 3B and S3A) but had different effects on CTxB-AF647 and Tfn-AF647 uptake (Figs. 2E, F, 3C, D, S3B and S3C). Again, no binding changes in any of the macromolecules listed above were observed among these transfections (Figs. 2D, E, F and S2C). Thus, the role of GRP75 in the promotion of αHS and CTxB uptake and in the inhibition of Tfn uptake is likely determined by the ATPaseD domain, but not by the SBD domain. 3.4. The lack of mitochondrial signal peptides compromises the endocytosis regulation activities of GRP75 The impact of the subcellular distributions (inside or outside of mitochondria) of GRP75 on its endocytosis regulation was then examined. Plasmid-transfected HeLa cells were subjected to test for analyzing the uptake of the indicated macromolecules. The expression of full-length wtGRP75 markedly increased the uptake of αHS-AF647 complex (average increases of 190% and of 180% were obtained with the FCM and confocal results, respectively), increased the uptake of CTxB-AF647 (average increases of 210%

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Fig. 2. Flow cytometry analysis of the uptake of αHS macromolecular complex with reference to the CIE marker CTxB and the CME marker Tfn in transfected cells. HeLa cells were first transfected with concentration-increased wtGRP75 plasmids following by GRP75-targeting RNAi (A, B, C), or transfected with the indicated plasmids as shown in Fig. 1(A) (D, E, F). The transfected cells were serum-starved for 30 min and then incubated with αHS-AF647 complex, Tfn-AF647 and CTxB-AF647 respectively on ice for binding-measurement after PBS/EDTA detachment, or incubated with these drugs at 37 °C for uptake-measurement after trypsinization. The cells were collected, rinsed and immediately analyzed alive via FACS. The expression levels of endogenous and EGFP fused-GRP75 proteins after transfection with increasing concentrations of wtGRP75 plasmids following RNAi were checked simultaneously via western blotting (A). Representative images of serial two-dimensional density plots obtained from three independent experiments are shown in (B). The uptake levels of αHS-AF647 complex, Tfn-AF647 and CTxB-AF647 in the transfected cells (validated by EGFP fluoresce, FL-1) were measured (FL-4) and are quantified in (C). The uptake and binding levels of these drugs in various transfections were measured and quantified as shown in (D), (E) and (F), respectively. The data collected include the following: means 7 SEM (error bars), n ¼3, Z 10000 EGFP positive cells counted per sample, and triplicate samples per transfection for each experiment. Statistically significant differences in comparison with the concentration-increased (C) or with the titer matched (D, E, F) pEGFP control are shown: ** P o0.01, * Po 0.05.

and of 195% were obtained with the FCM and confocal results, respectively) but decreased the uptake of Tfn-AF647 (average decreases of 53% and 56% were obtained with the FCM and confocal results, respectively) (Figs. 2D, E, F, 3B, C and D). In contrast,

ΔSGRP75 expression only slightly increased the uptake level of αHS-AF647 complex (average increases of 130% and 115% were obtained with the FCM and confocal results, respectively) and CTxB-AF647 (average increases of 135% and 120% were obtained

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Fig. 3. Confocal analysis of αHS macromolecular complex uptake with reference to the CIE marker CTxB and CME marker Tfn. HeLa cells were transfected with EGFP fused GRP75 plasmids and serum-starved as described above, then were respectively incubated in αHS AO4B08-AF647 complex, Tfn-AF647, and CTxB-AF647 at 37 °C. The cells were rinsed with PBS/1M NaCl, fixed and then visualized via confocal microscopy. Images are maximal projections of a z-series of fluorescence confocal slices through the entire cell volume. Representative images for three independent experiments are shown in (A). Scale bar, 20 mm. The αHS-AF647 complex, CTxB-AF647 and Tfn-AF647 uptake levels in EGFP recombinant plasmid-transfected cells in comparison with the pEGFP control levels are quantified in (B), (C) and (D), respectively. Uptake percentages are correspondingly corrected by the expression levels of EGFP recombinant proteins. The data collected include the following: means 7 SEM (error bars), n ¼3, Z 30 EGFP positive cells counted per transfection in each experiment. Statistically significant differences in relation to the pEGFP control are shown: ** P o0.01, * P o0.05.

with the FCM and confocal results, respectively) (Figs. 2D, E, 3B and C) but did not significantly decrease the Tfn-AF647 uptake levels (Figs. 2F and 3D). Similar differences in the effects on αHSAF647 and CTxB-AF647 uptake were also observed between ATPaseD and ΔS ATPaseD expression (Figs. 2D, E, 3B and C). However, no significant differences in the changes of αHS-AF647 complex, CTxB-AF647 and Tfn-AF647 uptake were found between SBD and ΔS SBD expression (Figs. 2D, E, F, 3B, C and D). Therefore, cells expressing ΔS GRP75 or ΔS ATPasD that lacked mitochondrial targeting sequences exhibited compromised uptakes of

macromolecular ligands compared with cells transfected with wtGRP75 or ATPaseD, suggesting that GRP75 trafficking through mitochondria is essential to endocytosis regulation. 3.5. Not only Cdc42 but also Rho A are activated by the expression of GRP75 or the ATPase domain It was found early on that GRP75 is bound with and involved in IL-1R1 internalization [8], and interleukin receptors are usually internalized via the RhoA-dependent CIE route [19,20]. Moreover,

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Fig. 4. The expression of wtGRP75 and ATPaseD stimulates RhoA and Cdc42 activation. HeLa cells were transfected with (A) GRP75-targeting siRNA pool or scramble siRNA, (B, C) EGFP-fused GRP75 plasmids with/without mitochondrial-targeting signal peptide and were serum-starved for 16 h, then the fractions of activated Cdc42 and RhoA were measured via GST pull down method as described in Section 2. Prime Abs with mouse anti-GRP75 (1:1000), rabbit anti-Cdc42 (1:200), mouse anti-RhoA (1:250), rabbit anti-b-actin (1:2000), and rabbit anti-GFP (1:2000) were respectively used for western blotting. Four independent experiments were performed and representative blot pictures for each of these experiments are shown in the left. The graphs shown in the right present the results from the quantitative analysis of the Cdc42 and RhoA activation levels determined via densitometry. Percentage changes were correspondingly corrected by the expression levels of EGFP-fused proteins. The data collected include the following: means 7 SEM (error bars), n ¼ 4. Statistically significant differences in relation to the appropriate controls (scramble siRNA or pEGFP) are shown: ** Po 0.01.

we previously showed that GRP75 is associated with αHS uptakeinduced Cdc42 activation and that the αHS uptake levels are reduced by half (compared with wtCdc42 transfection) in dominantnegative Cdc42 (T17N)-expressing cells [9]. To fully determine the role of GRP75 in Rho GTPase activation, HeLa cells were transfected with plasmids as noted above or with GRP75-targeting RNAi pool, and the activation levels of RhoA, Cdc42 and Rac1 were simultaneously compared. GRP75 knock-down by RNAi decreased the Cdc42 and RhoA activation levels to 55% and 35%, respectively, on average (Fig. 4A). In contrast, wtGRP75 expression induced nearly three times the increment in Cdc42 and RhoA activations. However, ΔS GRP75 expression did not induce any Cdc42 activation but a little increment in RhoA activation (Fig. 4B and C). Compared with transfection with full-length GRP75, the expression of ATPaseD increased the Cdc42 and RhoA activation levels to 410% and 300%, respectively, on average, whereas the expression of SBD or ΔS SBD reduced their activation levels to 70% or 40% and 45% or 20%, respectively, on average. It is noteworthy that ΔS ATPaseD expression did not significantly induce the activation of Cdc42 and RhoA (Fig. 4B and C). In addition, no Rac1 activation processes were detected after transfection of any of the indicated plasmids (data not shown here). Taken together, these results show that the expression of GRP75, particularly ATPaseD,

stimulates both RhoA and Cdc42 activation, whereas GRP75 knock-down and SBD expression limit RhoA and Cdc42 activation. 3.6. GRP75 acts upstream from Cdc42 and RhoA activation to regulate clathrin-independent endocytosis Though we previously found a fraction of co-localization between αHS and GRP75 in endocytic vesicles and co-localization between endocytic αHS and ectopically expressed Cdc42 [9], the relationship between GRP75 and Cdc42 had not been examined directly. Analyses of transfected HeLa cells conducted using a confocal microscope strikingly revealed variant co-localization patterns between endogenous GRP75 and ectopically expressed wtCdc42, Cdc42 (G12V) activated mutant, Cdc42 (T17N) dominant-negative mutant (Fig. S4A). Activated mutant Cdc42 (G12V) was predominantly co-localized with endogenous GRP75 on tubular mitochondria structures. Only a small fraction of wtCdc42 was clearly co-localized with endogenous GRP75 in mitochondria of the perinuclear area. In contrast, dominant-negative Cdc42 (T17N) mutant was seldom co-localized with endogenous GRP75 (Fig. S4A). However, such results were not found in the co-immunoprecipitation of activated mutant Cdc42 by EGFP-GRP75 (Fig. S4C). In addition, we did not find interaction binding between

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Fig. 5. GRP75 acts upstream from RhoA and Cdc42 activation in CIE regulation. HeLa cells were transfected first with Cdc42-targeting siRNA pool (A), Cdc42(G12V) activated mutant (CA-Cdc42) (B), or RhoA-targeting siRNA pool (C) respectively, and then followed with GRP75 plasmid overexpression (w GRP75 OE) or with its RNAi knock-down (w GRP75 RNAi) or without further treatments (wo). Negative controls are scramble siRNA transfection followed by the pEGFP transfection (A, C), or the pCMV5 transfection followed by scramble siRNA & pEGFP transfection (B). The corresponding cell lysates were analyzed via western blotting using mouse anti-GRP75 Ab (1:1000), rabbit antiCdc42 Ab (1:200), mouse anti-RhoA Ab (1:250) and rabbit anti-FLAG Ab (1:2000). The αHS-AF647 uptake in the transfected cells based on staining using corresponding Ab combinations was measured by flow cytometry (D) and confocal microscopy (E) as described in Section 2. Rabbit anti-GRP75 (1:100), goat anti-FLAG (1:100), anti-rabbit AbAF488 (1:1000) and anti-goat Ab-AF546 (1:1000) were used together in co-staining to determine the overexpression/interference levels of GRP75 and/or Cdc42. The αHSAF647 uptakes values for the different treatments relative to the control levels (Lipo 2000 or Lipo RNAiMAX) are quantified in (D) and (F). The data used include the following: means 7 SEM (error bars), n ¼3, with Z 10000 cells counted per sample in the flow cytometry experiments and with Z30 targeted cells (arrows, GRP75 OE. arrowhead, GRP75 RNAi) counted per transfection in the confocal experiments. Scale bar, 20 mm. Statistically significant differences in relation to the respective controls are shown: ** P o 0.01.

activated RhoA (EGF stimulated) and endogenous GRP75 in copulldown assay (Fig. S4B). To fully explore the relationship between GRP75 and Cdc42/ RhoA in the CIE pathway, HeLa cells were transfected with Cdc42/ RhoA siRNA pool and then subjected to GRP75 overexpression or knock-down (Figs. 5A, C and 6), or were transfected with Cdc42 (G12V) activated mutant and then subjected to GRP75 overexpression or knock-down (Fig. 5B). Flow cytometry and confocal microscope analyses showed that Cdc42 and RhoA knock-down significantly decreased αHS-AF647 complex uptake (Fig. 5D, S7), whereas Cdc42 (G12V) activated mutant expression markedly increased αHS-AF647 complex uptake (Figs. 5D, E and F). The decrease in the uptake of αHS-AF647 complex induced by Cdc42 or

RhoA knock-down was aggravated by GRP75 knock-down, whereas wtGRP75 expression completely reversed this effect (Fig. 5D). In contrast, increased αHS-AF647 complex uptake induced by Cdc42 activated mutant was synergistically promoted by wtGRP75 expression (Fig. 5F). However, this increase in αHSAF647 complex uptake was not significantly affected by GRP75 knock-down (Fig. 5D, E and F). Similar changes in αHS-AF647 uptake were observed after combined modulation of the expression of GRP75 and Cdc42 or the expression of GRP75 and RhoA (Fig. S5). In addition, simultaneous knock-down of Cdc42 and RhoA abrogated the effect of GRP75 expression on uptake promotions of aHS complex and CTxb (Fig. 6). Compared with the results in Fig. 5(D), a compensatory relationship between Cdc42

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Fig. 6. Simultaneous knock-down of Cdc42 and RhoA abrogate the effect of GRP75 overexpression on CIE promotion. HeLa cells were reverse-transfected with Cdc42targeting siRNA pool for 6 h first, then forward-transfected with RhoA RNAi pool for 6 h after 8 h growth. The double transient-transfected cells were digested and reseeded with reverse-transfection of wtGRP75 plasmid (w GRP75 OE) or GRP75 RNAi pool (w GRP75 RNAi) overnight, or without further treatments (WO). Negative controls are transfections with scramble siRNA pools followed by GRP75 overexpression or RNAi. The efficiency of knockdown and overexpression of interest proteins were checked (rabbit anti-Cdc42, 1:200; mouse anti-RhoA, mouse anti-GRP75, 1:1000; 1:250, rabbit anti-GFP, 1:2000, and rabbit anti-b-actin, 1:2000) and representative results of western blotting are shown (A). The uptakes values of αHS-AF647 complex, CTxB-AF647 and Tfn-AF647 for different treatments relative to the control levels are quantified in (B, C, D). The data used include the following: means 7 SEM (error bars), n ¼ 3, with Z 10000 cells counted per sample in flow cytometry experiments. Statistically significant differences in relation to the control are shown: ** Po 0.01.

and RhoA may exist in GRP75 mediated endocytosis regulation. All these results indicated that neither Cdc42 nor RhoA binds directly to GRP75, and that concurrent RhoA and Cdc42 activation constitute downstream signaling events in GRP75-regulated CIE pathways. 3.7. Expression of GRP75 and its ATPase domain induce actin cytoskeleton reorganization Because members of Rho GTPase have long been known to be extensively involved in endocytosis regulation and in controlling cytoskeleton dynamics [21], we further examined the effects of GRP75 and its limited-domain plasmids expression on actin cytoskeleton organization via phalloidin-Rhodamine labeling. HeLa cells transfected with the control pEGFP plasmid were elongated with few actin microfilament bundles forming thin peripheral bands and fewer stress fibers, whereas wtGRP75-and ATPaseDtransfected cells were polygonal with increased polymerized actin forming a wider cortical actin layer of high stress fiber accumulation (Fig. 7A, arrowheads, Fig. 7D) and numerous filopodia (Fig. 7A, arrows, Fig. 7B). The filopodia in the ATPaseD expressing cells were significantly shorter than those found after wtGRP75 transfection (Fig. 7A, arrows). In contrast, ΔSGRP75-and ΔSATPaseD-transfected cells showed intermediate levels of cortical

actin filament accumulation (Fig. 7A, arrowheads, Fig. 7D) but rarely showed filopodia (Fig. 7B). As shown in Fig. 7C, lamellipodia formation (asterisk) was largely absent after all construct transfections and was only observed in ΔS GRP75-transfected cells. It is noteworthy that little or no formation of stress fibers, filopodia and lamellipodia was observed in the SBD- and ΔS SBD-transfected cells (Fig. 7B, C and D). These results show that GRP75 or ATPaseD expression primarily results in enhanced stress fiber formation and significantly increases filopodia presentation, strongly suggesting remarkable actin cytoskeletal reorganization. Most CIE pathways share virtually the same molecular machinery (particularly actin cytoskeleton system) to affect actual endocytic processes [1]. To examine whether the promotion of CIE induced by GRP75 was because of enhanced actin dynamics, we used pharmacological inhibitor of actin polymerization. Although variable effects on CME inhibition had been observed when actin polymerization inhibitors were used at higher concentration or for longer incubation times [20], we found 5 min incubation with Lat A at 5 μM could reach a distinct CIE inhibition in HeLa cells (Fig. S6). Treatment of wtGRP75-transfected HeLa cells with Lat A markedly blocked the uptake of aHS complex and CTxb to a similar extent, at both 1 h (data not shown) as well as 30 min, while the uptake of Tfn (at 15 min) was relatively unaffected (Fig. 7E, F and G); Similar to the perturbation on wtGRP75-induced endocytosis,

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Fig. 7. The expression of wtGRP75 and ATPaseD induces actin cytoskeleton reorganization and the inhibition of actin polymerization blocks CIE. HeLa cells were transfected with indicated plasmids as shown in Fig. 1, reseeded in poly-D-lysine pre-coated chamber slides, serum-free starved, then were fixed, permeabilized and co-stained with rabbit anti-GFP Ab (1:1000, followed by anti-rabbit Ab-AF488) and Rhodamine-conjugated phalloidin (100 nM) for detection of polymerized actin. Representative confocal images for three independent co-staining experiments are shown in (A). Scale bar, 20 mm. Morphometric analyses of the extent of filopodia, lamellipodia and stress fiber formation in the transfected cells are shown in (B), (C) and (D), respectively. The transfected cells starved in parallel were subjected to Lat A treatment before the flow cytometry based uptake-checking of indicated macromolecular drugs as described in Section 2. The uptake levels of αHS-AF647 complex, Tfn-AF647 and CTxB-AF647 in transfected cells are respectively quantified in (E), (F) and (G), expressed relative to that measured in un-transfected cells (value setting to 1). The data used include the following: means 7SEM (error bars), n ¼ 3, with 4 60 cells counted per transfection in confocal experiments and Z 10000 cells counted per sample in flow cytometry experiments. Statistically significant differences in relation to the respective controls are shown: ** P o 0.01, * P o0.05.

treatment of ΔS GRP75-transfected cells with Lat A also decreased the uptake of aHS complex and CTxb but did not affect Tfn uptake (Fig. 7E, F and G), indicating that the effects of actin perturbation on CIE may be more severe than that on CME. These results demonstrated that the CIE promotion by GRP75 is strongly modulated by the dynamic status of the actin cytoskeleton.

4. Discussion Although the reduction in the dynamic trafficking of mitochondrial protein into endocytic vesicles is difficult to realize, signal peptide-directed protein subcellular transport based on overexpression methodology can be feasibly employed to mimic the enrichment process and thereby uncover gain-of-function properties. In the present study, we demonstrate that the uptake of HSPG-dependent macromolecular complex occurs through CIE pathway mediated by the concurrent downstream activation of RhoA and Cdc42 GTPases, which are regulated by upstream GRP75. This endocytosis regulation is largely mediated by the subcellular location of GRP75 protein in mitochondria and is essentially determined by the ATPaseD characteristics. The overexpression of GRP75 or its ATPaseD in mitochondria stimulates RhoA and Cdc42 activation and enhances the formation of stress fibers and

filopodia, collectively resulting in the promotion of CIE but the inhibition of CME. GRP75, which is mainly found in mitochondria, is a 679-aminoacids-long, heat-uninducible member of the Hsp70 protein family. In general, HSP70 heat shock proteins bind to unfolded proteins via their C-terminal SBD, and their chaperone activities require ATP hydrolysis executed by their N-terminal ATPaseD [10]. In the chaperone/Tom70 system of mitochondrial protein delivery, the ATPase hydrolysis cycling of a multichaperone complex (including Hsp90 and Hsp70) drives preproteins transfer from the mitochondrial Tom70 complex through the import pore to the TIM (inner membrane import machinery) [22]. Although specific molecules binding to GRP75 during the CIE process have not yet been identified, in this study, we show that the ATPaseD, similarly to intact GRP75, but not the SBD, promotes αHS and CTxB uptakes but inhibits Tfn uptake (Figs. 2D, E, F and 3). These results are in line with two previous findings: 1) HSP90-mediated intercellular vesicle transport, VSVG trafficking between Golgi stacks is dependent on the ATPaseD of HSP90; and 2) in immune attacked cells, the caveolin- and dynamin-dependent endocytosis of MAC is regulated by GRP75 through its ATPaseD but not through SBD [12]. All of these results reveal the decisive contributions of ATPaseD to the endocytosis regulation function of GRP75. No studies have previously shown any functional connection

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between an HSP70 chaperone and Rho GTPase. The protein that may be related (with only referential significance) was HSP90, which contributes to protein trafficking by forming a complex with GDI to direct the intracellular recycling of Rab GTPase [23]. In this study, we found that GRP75 expression significantly stimulates RhoA and Cdc42 activation, whereas GRP75 knock-down limits their activation (Fig. 4). Furthermore, we found that GRP75 acts upstream of the concurrent activation of RhoA and Cdc42 in this CIE pathway (Figs. 5, 6). To the best of our knowledge, this study provides the first demonstration that the HSP70 chaperone acts as an upstream regulator of Rho GTPase. Whether the mechanism underlying the effects of GRP75 on RhoA and Cdc42 activation is similar to that involved in the Hsp90 regulation of GDIdependent Rab recycling (i.e., via binding with Rho GDI) remains to be elucidated further. Strikingly, an unexpected finding of the present study is that both RhoA and Cdc42 are concurrently activated by wtGRP75 expression (Fig. 4). Although specific requirements for a given CIE pathway have not been rigorously established, an increasing body of evidence suggests that RhoA activation serves as the driving machinery behind the uptake of cytokine receptors and that Cdc42 activation serves as the driving machinery behind the endocytosis of CTxB and GPI-APs [19]. Because glypicans form GPI-APs on the cell surface and GRP75 is involved in IL receptors internalization and αHS-F complex uptake [8,9], the concurrent activation of RhoA and Cdc42 by wtGRP75 expression found in the present study is reasonable. In addition, it is noteworthy that, similarly to Cdc42 knock-down, RhoA knock-down also significantly decreased the uptake of αHS-F complex (Fig. 5D). Therefore, cell surface GPI-APs crosslinked by αHS Ab (AO4B08) may have been internalized by mixed CIE pathways. We previously reported that endogenous GRP75 is markedly enriched in endocytic vesicles while being localized on the HeLa cell surface [9]. In this study, we showed that the main fraction of expressed wtGRP75 becomes trans-localized into the mitochondrial membrane structure (Fig. 1C). These observations are consistent with previous observations that 70-kDa HSPs (including GRP75) are selectively localized in detergent-resistant membrane fractions in several tissue cells [24–28], thus suggesting that GRP75-membrane enrichment or cell surface translocation may exist to maintain the stability of lipid raft-associated structures. GPI-APs, including glypicans, are always found on the cell surface in cholesterol-enriched nanoscale clusters. These clusters have been found to act as sorting platform for the selective uptake of GPI-APs via Cdc42-activation dependent CIE pathway [29]. In addition, most Rho GTPases are usually recruited and activated by the Dbl family GEFs (guanine nucleotide exchange factors) positioned very close to cell membranes, and post-translational modifications (e.g., protein prenylation or palmitoylation) often enhance their bindings with certain components in cell membrane while determining their localization in specific membrane compartments [21]. When considering these facts together, it is logical to assume the presence of intermolecular interaction or binding between GRP75, HSPG (glypican in particular) and Cdc42 on cell surfaces or in endocytic vesicles. However, only small levels of colocalization between GRP75 and HSPG and between GRP75 and Rho GTPase were observed in the confocal assays [9]. Co-localization between GRP75 and the lipid raft marker CTxB and molecular interactions between GRP75 and Rho GTPase or HSPG have not been found in any of our previous studies [9] (Figs. 3, 5, S2 and S4). In contrast, after translocation to the cell surface, another member of the HSP70 protein family, GRP78, which is primarily localized in the endoplasmic reticulum, was frequently found to act as a binding receptor for the lipid raft- or caveolae-dependent endocytosis of coxsackievirus A9, dengue virus serotype 2, activated 2-macroglobulin and dentin matrix proteins [4–6]. It is widely recognized that HSPG serves as a co-receptor for these

types of viruses, and chondroitin sulfate chains are known to attach to bone dentin matrix proteins [30]. It was shown early on that Hsp70s are prone to binding to hydrophobic or basic residuesenriched peptides [31], and the endocytosis ligand used in this study, AO4B08 αHS Ab (2-O-sulfation HS epitope), also showed such characteristic sequence [16]. In addition, HSP70 protein was recently found to preferentially bind to and form a complex with heparan sulfate, which often contains 2-O-sulfated iduronic acid residue [32]. All of these findings encourage us to adopt novel methodologies for identifying specific binders to GRP75 in endocytosis. Although several chaperones have been suggested to regulate the cytoskeleton system, the only one that has been validated and that is known to be involved in membrane trafficking is HSP27. HSP27 has been found to exhibit increasing F-actin concentrations in the CHO cell cortex, and its overexpression enhances pinocytosis [33]. Its role on the induction of actin polymerization was further found to accompany decelerated Tfn endocytosis in CCL39 cells [34] and enhanced exosome uptake in HUVEC cells [35]. In the present study, we provide the first demonstration that expression of GRP75 and its ATPaseD enhances stress fiber formation and thereby increase the quantities of filopodia in HeLa cells (Fig. 7). These enhanced actin polymerizations occur alongside the promotion of uptake of αHS and CTxB and the inhibition of Tfn uptake (Figs. 2, 3 and S3). These results indicate that in comparison with HSP27, GRP75 exerts a similar effect on stabilizing the assembly of the actin cytoskeleton while playing a major role in endocytosis regulation. In fact, Hsp27 is an actin-binding protein that is frequently co-localized with F-actin in non-stimulated neutrophils [36]. However, we did not observe such co-localization between GRP75 and F-actin (Fig. 7). In general, actin plays multiple roles in endocytosis modulation by facilitating membrane deformation and vesicle movement within the cells. Founding members of the GTPase Rho family (RhoA, Cdc42 and Rac1), when activated, always interact with downstream effectors that collectively trigger actin cytoskeleton reorganization, which is further maintained by dynamic Rho GTPase activation near the membrane structure [21,29,37]. Thus, it is logical to deduce from the present results that the expression of GRP75 or of its ATPaseD in mitochondria stimulates RhoA and Cdc42 concurrent activation, in turn enhancing actin cytoskeleton reorganization and upregulating CIE (Fig. 8). Notably, the present results differ from previous findings, which demonstrated that another chaperone protein, Hsp90, downregulates Rho GTPase activity [38] while mainly facilitating Rab-GTPase recycling [23]. Because HSP70 and HSP90 play different but cooperative roles in cytoskeleton assembly and RhoGEF activity maintenance [39,40] and because bioinformatics methods predict that many components of the endocytosis pathway may serve as downstream targets of Hsp90 [41], it is naturally speculated that GRP75 may function in or interact physically with some unknown factors (e.g., DBL GEF family members), potentially resulting in the concurrent activation of both RhoA and Cdc42 in CIE (Fig. 8). This hypothesis is currently being investigated in our laboratory. In summary, this study shows that GRP75 induces concurrent downstream activation of RhoA and Cdc42, enhances the formation of stress fibers and filopodia, and promotes CIE but inhibit CME. All of these processes are mediated by its mitochondrial location and ATPase domain. The present results strongly suggest that GRP75 subcellular translocation/trafficking/enrichment upregulates CIE through actin cytoskeleton reorganization mechanisms mediated by the concurrent activation of Cdc42 and RhoA. These findings serve as an essential step toward understanding complex chaperone signaling in the regulation of cell endocytosis pathways and toward uncovering a potentially useful macromolecular delivery route in cells.

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Fig. 8. A schematic model of the regulatory function of GRP75 trafficking in CIE. HS 2-O-sulfation epitope-specific anti-HSPG Ab AO4B08 (αHS), co-localized with the lipid raft marker CTxB, is internalized by CIE. αHS internalization induces mitochondrial chaperone protein GRP75 subcellular translocation/trafficking/enrichment to the cell surface or to membranous organelles via an unknown mechanism, stimulating RhoA and Cdc42 activation and markedly inducing actin cytoskeleton reorganization. All of these changes significantly promote CIE while simultaneously inhibiting CME.

Disclosure of potential conflicts of interest The authors declare no conflicts of interest.

Author contributions Sihe Zhang initiated the idea and the working approaches, supervised the research and wrote the manuscript. Hang Chen performed most of the experiments and data organization. Toin H. van Kuppervelt and Mattias Belting provided important materials and technical supports. Other authors performed some of the experiments.

Acknowledgments We are grateful to Prof. Kenji Fukasawa, Prof. Takaya Satoh and Prof. Alan Hall for providing valuable plasmids. This work was supported by the National Natural Science Foundation of China (Nos. 81373318, 30700829), the Specialized Research Fund for Doctoral Program of Higher Education of China (No. 20130031120037), the Natural Science Foundation of Tianjin City (No. 13JCYBJC21000) and the Open Fund of State Key Laboratory of Medicinal Chemical Biology (Nankai University) (No. 20130575).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2016.04.009.

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