Cytoskeletal Control of Nuclear Morphology and Chromatin Organization

    Cytoskeletal control of nuclear morphology and chromatin organization Nisha M. Ramdas, G.V. Shivashankar PII: DOI: Reference:

S0022-2836(14)00495-1 doi: 10.1016/j.jmb.2014.09.008 YJMBI 64563

To appear in:

Journal of Molecular Biology

Received date: Revised date: Accepted date:

9 April 2014 3 September 2014 6 September 2014

Please cite this article as: Ramdas, N.M. & Shivashankar, G.V., Cytoskeletal control of nuclear morphology and chromatin organization, Journal of Molecular Biology (2014), doi: 10.1016/j.jmb.2014.09.008

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ACCEPTED MANUSCRIPT Title

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Cytoskeletal control of nuclear morphology and chromatin organization

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Author Affiliations

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Nisha M. Ramdas1 and G.V. Shivashankar2,*

1National Centre for Biological Sciences, TIFR, Bangalore, India

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2Mechanobiology Institute and Department of Biological Sciences, NUS, Singapore

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*Corresponding author

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Corresponding Author

G.V. Shivashankar, Mechanobiology Institute, National University of Singapore, T-Lab #05-

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01, 5A Engineering Drive 1, Singapore 117411. Email: [email protected]

ACCEPTED MANUSCRIPT Abstract The nucleus is sculpted towards various morphologies during cellular differentiation and

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development. Alterations in nuclear shape often result in changes to chromatin organization and

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genome function. This is thought to be reflective of its role as a cellular mechanotransducer.

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Recent evidence has highlighted the importance of cytoskeletal organization in defining how nuclear morphology regulates chromatin dynamics. However the mechanisms underlying

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cytoskeletal control of chromatin remodeling are not well understood. We demonstrate here, the

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differential influence of perinuclear actin- and microtubule-driven assemblies on nuclear architecture using pharmacological inhibitors and targeted RNAi knockdown of cytoskeleton

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components in Drosophila cells. We find evidence that the loss of perinuclear actin assembly

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results in baso-lateral enhancement of microtubule organization and this is reflected functionally by enhanced nuclear dynamics. Cytoskeleton reorganization leads to nuclear lamina deformation

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which influences heterochromatin localization and core histone protein mobility. We also show that modulations in actin-microtubule assembly results in differential gene expression patterns. Taken together, we suggest that perinuclear actin and baso-lateral microtubule organization exerts mechanical control on nuclear morphology and chromatin dynamics.

ACCEPTED MANUSCRIPT Chromatin compaction involves the condensation of DNA into a compact structure that fits within the confines of the nucleus. This compaction, which is largely mediated by histone 1,2

is known to be a significant factor in the regulation of gene expression

3–5

. Evidence

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proteins

determinant of genome organization

6–8

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suggests that the association of chromatin with the nuclear periphery could serve as a prime . The mechanical integrity of the nuclear periphery is

11,12

as well as multiple disease states

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chromatin plasticity

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defined by the nuclear lamina 9,10 where deficiencies in specific components result in nuclear and 13–15

. Recent studies indicate that the

nuclear lamina is coupled to the cytoskeleton via LINC (Linker of Nucleoskeleton and

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Cytoskeleton) complexes present on the nuclear envelope

16–18

. This structural link between the

cytoskeleton and nucleus, referred to as the nucleo-cytoskeleton, enables cell surface forces and

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substrate rigidity to influence nuclear morphology 19–21. It also enables the transduction of forces

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to the nucleus which alter chromosome movement and nuclei positioning 22–24. Thus, the nucleus

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is mechanically influenced by nucleo-cytoskeletal assemblies located within the cytoplasm. Studies on nuclear morphology have demonstrated that the actin filament network plays a role in establishing and maintaining the form and orientation of nuclei

25–27

while the associated

actomyosin tension maintains cellular stiffness and nuclear prestress

28–31

. Also crucial in these

processes are microtubule assemblies, which are intricately and dynamically linked to actin and have been shown to bear large compressive stresses within cells

32

. Microtubules are rigid

cytoskeletal filaments that are more resistant to bending than actin filaments

33

, and coordinate

with actin to regulate actomyosin contractility 34,35, cell movement and morphogenesis 36. Recent studies have also provided evidence of their mechanical influence on the nuclear envelope and chromosome dynamics

37–39

, however, the integrated coupling between actin and microtubule

networks in modulating nuclear morphology and chromatin function are not well understood.

ACCEPTED MANUSCRIPT Here, we address this question by using Drosophila cells to assess the role of altered cytoskeletal assemblies on nuclear form, and dissect their influence on chromatin organization and dynamics.

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The actin and microtubule cytoskeleton exert differential control on nuclear morphology

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Cytoskeletal architecture was perturbed using pharmacological agents that either depolymerized

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or stabilized components of the perinuclear actin-microtubule network, which altered the force 27,40

. Illustrated in Fig 1A and 1B are

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balance between the cytoskeleton and the nucleus

representative images of Hoechst stained Drosophila S2R+ cells where changes in the nuclear

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morphology have accompanied the coordinated architectural modulation of phalloidin-stained actin and α-tubulin stained microtubules. Control cells displayed a large flattened cell body with

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actin assembled largely at the basal periphery, and with a tightly organized microtubule network

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basally-centrally-apically surrounding the flattened nucleus (Fig 1A, 1B). Following perturbation

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of the actin cytoskeleton using latrunculin A (LatA), which inhibits actin polymerization loss in actin compressive forces long processes

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19

41

, the

caused the cell body to retract as the microtubules extended

. Recent studies have shown that the organization of lateral microtubules

together with fluctuations in the forces they transduce, are influenced by their surrounding cytoskeleton assemblies

43,44

. As shown in Fig 1E, the alteration in microtubule organization

upon actin perturbation resulted in nuclear elongation, which was quantified by an extended height and reduced nuclear projected area (PA). However, microtubule disruption using nocodazole (Noc)

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, resulted in further flattening of the nucleus

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(Fig 1A,1E). In contrast,

stabilization of microtubule assembly using paclitaxel (Pac), an anti-cancer drug

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, resulted in

enhanced microtubule organization accompanied by concomitant nuclear elongation (Fig 1A, 1E). Combining microtubule stabilization with perturbation to actin assembly (Pac+LatA), lead to further nuclear elongation (Fig 1A, 1E). The enhancement in lateral microtubule assembly due

ACCEPTED MANUSCRIPT to actin perturbation and microtubule stabilization

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, and asymmetry in its organization around

the nucleus, is demonstrated in Fig 1B which depicts the cellular organization at the central plane

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of the cell. This modulation in nuclear-microtubule architecture demonstrated the influence of

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the microtubule organization on nuclear morphology.

level of the nuclear lamina architecture

40,48

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We next examined whether changes in the cytoskeletal force-balance, would be mirrored at the . The organization of the immunostained nuclear

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lamin protein Dm0 (Fig 1C) was quantified via an assessment of its shape, using the nuclear

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shape index (SI) (Fig 1F). In flattened control nuclei, nuclear lamin was tightly arranged at the periphery of the organelle (Fig 1C). Following LatA treatment however, a loss of perinuclear

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actin compressive force lead to a smaller and rounded lamin organization that accompanied

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nuclear elongation. Futhermore, microtubule disruption (Noc) resulted in a flattened lamin organization. In contrast, enhanced microtubule forces (Pac and Pac+LatA) revealed marked

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lamin deformation and concomitant anisotropic nuclear shapes (Fig 1C, 1F). This provided further evidence that lateral microtubule organization plays a role in the modulation of nuclear morphology. Furthermore, 3D nuclear lamin reconstructions showing changes in nuclear morphology (Fig 1D) provided illustrated evidence of altered mechanics at the nuclear periphery and lead us to suggest that nuclear elongation and deformation is a combined consequence of perinuclear actin perturbation and altered microtubule organization. Additionally, a change in the localization of the nucleus within the cell is associated with alterations in nuclear form, and this can be assessed by visualizing the cellular organization at the central plane (Fig 1B). A loss of symmetry in the position of the nucleus within the cell can be quantified using the nuclear position index (PI). Using this method, an enhanced offset from the cell centroid was noted following alterations to the balance of cytoskeletal forces (Fig 1F).

Taken together, our

ACCEPTED MANUSCRIPT observations suggest that coordination between perinuclear actin and lateral microtubule cytoskeleton assemblies results in a differential force balance that modulates nuclear morphology

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and positioning.

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Coordinated cytoskeleton modulation of nuclear morphology

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To further probe the differential influence actin-microtubule networks have on nuclear

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morphology, while ensuring minimal perturbation to cellular structure and function, we employed a limited RNAi based screening technique. Here, a biased list of target genes was

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established based on their role in the integrated nucleo-cytoskeleton network. These targets were then screened to establish their influence on the shape and size of the nucleus.

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Fig 2A shows representative images of Hoechst stained nuclei in control cells, along with the distinctive multi-nucleated phenotype of RNAi positive control cells (Pav (CG1258)). Analysis

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of the nuclear morphologies that emerged after 5-day RNAi depletion revealed variability in nuclear metrics even across control wells. For this reason, we employed a method of phenotype detection that evaluated differences in the distribution tails of target genes as compared to control genes. These differences were quantified by establishing the log mean fold ratio of changes in the upper/lower bounds of the morphometric parameters (Fig 2B). RNAi targeting of cytoplasmic and nuclear related proteins resulted in altered nuclear shape and size (Fig 2C). Quantitative assessment of morphometric changes to nuclei following RNAi screening revealed a phenotypic trend that was in contrast to control nuclei. As illustrated by the representative images in Fig 2D, targeting of actin-components resulted in elongated nuclei while targeting of microtubule-components resulted in flattened nuclei. These results are summarized by a phenotypic profile heatmap (Fig 2E) which provides further evidence that elongated nuclei

ACCEPTED MANUSCRIPT (which are defined by a smaller nuclear PA and higher SI) result from the targeting of genes involved in actomyosin contractility. In contrast, large flattened nuclei (with a larger nuclear PA

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and lowered SI) result from targeting genes involved in microtubule assembly (Fig 2E). These

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phenotypes are consistent with enhanced lateral microtubule forces and enhanced actin forces, respectively. The observed alterations to nuclear shape and size via targeted cytoskeleton

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perturbation further reinforces our hypothesis that perinuclear actin-microtubule assemblies play

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a coordinated role in the establishment and maintenance of nuclear morphology.

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Alteration to nuclear and chromatin dynamics by actin-microtubule assemblies Having established the importance of perinuclear actin-microtubule assemblies in the

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maintenance of force-balance and nuclear morphology, we wished to determine whether the

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spatial organization of these structural assemblies also had an impact on nuclear and chromatin

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dynamics.

To investigate this, actin and microtubule linker proteins were targeted by RNAi, and the spatial organization of microtubules, which balances the actin architecture

27

, was assessed (Fig 3A).

Following knockdown of two microtubule-associated linker proteins, specifically the SUNdomain protein Klaroid

49

(CG44154) and the KASH-domain protein Klarsicht

50

(CG17046),

flattened cells and nuclei were observed. In this case, a tight spatial link between the cell and the nucleus (cell-nucleus anchorage), was maintained, ensuring only a slight displacement of the nucleus from the base of the cell. In contrast, knockdown of actin-associated linker proteins, including the SUN-domain protein Spag4 51 (CG6589) and the KASH-domain protein Msp300 52 (CG42768), resulted in the enhancement of basal microtubule organization (Fig 3A), and a disrupted spatial link between the cell and the nucleus. As shown in Fig 3B, the offset in nuclear

ACCEPTED MANUSCRIPT anchorage from the cell base was a significant consequence in the knockdown of perinuclear actin-associated linker proteins. Taken together, these results suggest that a structural balance

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between perinuclear actin and lateral/basal microtubules provide a force-balance which

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influences the morphology of the nucleus.

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Earlier studies have demonstrated that, by serving as load bearing structures in interphase cells , functionally repressive heterochromatin plays a mechanical role in the establishment and

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maintenance of nuclear morphology. We therefore asked whether cytoskeletal assemblies alter

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the spatial organization of heterochromatin structures. This was achieved in both control and actin-associated linker Msp300 RNAi targeted cells, by immunostaining HP1α heterochromatin

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and compiling images along the height of the nucleus, from the basal to the apical plane.

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As reflected by the color-coded intensity images and z-outlines of the nucleus (Fig 3C),

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heterochromatin organization in flattened nuclei shows minimal change. However, in cells where the nucleus is deformed along its height by lateral microtubule assembly, heterochromatin organization also exhibits significant variation. This was seen in the Msp300 perturbed nuclei by the larger heterochromatin shape index (SI), which quantified the variation in nuclear shape (as seen from immunostained heterochromatin images) along the height of the nucleus (Fig 3D). It was also evident by the deformations in the outlines of the nucleus (marked in green color), as compiled from the projected z-outline images along the nuclear height (Fig 3C). Our results further indicate that as a consequence, the spatial localization of the more condensed load bearing heterochromatin structures is altered along the height of these deformed nuclei. This is distinguished in the color-coded intensity images by the altered localization of the high-intensity heterochromatin regions (marked in red color), in contrast to their z-outlines, and quantified by the larger heterochromatin position index (PI) (Fig 3C, 3D).

ACCEPTED MANUSCRIPT To further establish whether changes in the spatial organization of load bearing heterochromatin structures, and perturbations to nucleo-cytoskeleton mechanics, altered chromatin organization,

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we evaluated changes in chromatin boundary dynamics via time-lapse imaging of H2B-EGFP

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labeled nuclei, in cytoskeleton perturbed cells (Fig 3E). Here, chromatin boundary fluctuation was measured over time and calculated against its center of mass via a quantitative assessment

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of the mean squared fluctuation 54 Σ (δ rθ2)/N, of the chromatin boundary, where rθ captured the

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radial distance of the nuclear periphery from the centroid at each of the different angles θ. The organization of the actin and microtubule cytoskeletal networks was perturbed and assayed after

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knocking down structural components, and functionally integral proteins, of each network. Specifically, the actin structural protein Act5c (CG4027), the focal adhesion protein, vinculin,

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(Vinc (CG3299)) and the actin-associated nesprin homolog, Msp300 (CG42768) were targeted

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for their influence on the actin network, while the β tubulin protein, βTub56D (CG9277) and

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kinesin heavy chain protein, Khc (CG7765) were targeted for their alteration to microtubule organization and motor assembly.

The perinuclear actin cytoskeleton exerts compressive forces that maintain the rigidity of the nucleus and help anchor it within the cell. Because of this, perturbation to the organization of the microtubule network did not induce fluctuations in the chromatin boundaries beyond what was observed in control cells, which was defined as minor fluctuations (Fig 3E). However, perturbations to the perinuclear actin cytoskeleton linkers disrupted nuclear anchorage, and consequently resulted in chromatin boundary fluctuation as quantified by enhanced mean squared fluctuations. Our findings are consistent with earlier reports demonstrating the influence of actin in constraining nuclear orientation

25

and the influence of microtubule forces on nuclear

envelope dynamics 55. Together, these results demonstrate the importance of perinuclear actin in

ACCEPTED MANUSCRIPT nuclear anchorage; with perturbations to its assembly together with enhanced lateral microtubule assembly, inducing fluctuations in nuclear dynamics.

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To further evaluate whether an alteration in nuclear morphology affects chromatin organization,

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we conducted fluorescence recovery after photobleaching (FRAP) experiments. A dissection in

dynamics of chromatin organization

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the mobility of core histones by FRAP analysis provides insights into differences in the . Quantitative analysis of the fluorescence recovery time

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of the core histone H2B-EGFP, revealed enhanced histone mobility following RNAi targeting of

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structural components of the actin network, and its associated linkers (Fig 3F). We note however, that given the enhanced nuclear mobility in actin perturbed nuclei as shown in Fig 3E, the

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increased mobility of the whole nucleus through the imaging plane during FRAP could also

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contribute to the rapid core histone recovery in the actin perturbed nuclei. In contrast, the mobility of core histones following knockdown of microtubule associated proteins, were similar

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to that of the control. Therefore, our results indicate that changes in chromatin organization occur concomitantly with changes in the shape and size of nuclei, specifically from a flattened to an elongated form. This is consistent with reports that suggest increased chromatin condensation is associated with enhanced organization of the actin cytoskeleton

11,25

. Furthermore, our findings

indicate that changes in nuclear morphology, which occur as a result of the coordinated organization of the perinuclear actin and microtubule cytoskeletal networks, could result in functional changes that are reflected by enhanced nuclear boundary dynamics as well as core histone mobility. In summary, the differential modulation of perinuclear actin-mediated contractility and basolateral microtubule assembly influences nuclear dynamics from several perspectives. Not only does it impact the establishment and maintenance of nuclear morphology but it also modulates

ACCEPTED MANUSCRIPT the localization of heterochromatin, and influences chromatin dynamics and histone mobility. Furthermore, perturbation of the perinuclear actin network alters the spatial links that anchor the

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nucleus to the base of the cell.

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Cytoskeleton modulation of nuclear architecture imposes changes in gene expression

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To further understand the influence cytoskeleton modulated nuclear morphology has on gene

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expression, a whole genome transcriptome analysis was performed using microarray. Specific components of the actin and microtubule networks were selected for RNAi targeting based on

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their role in linking the cytoskeleton to the nucleus; targets included the microtubule linker SUNprotein Klaroid, the actin linker KASH-protein Msp300 and the nuclear lamina scaffold protein,

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Lamin. Representative images depicting an altered nuclear morphology, which resulted from the

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genetic perturbation of the selected genes (Fig 4A), as well as a quantitative assessment of these

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altered nuclear morphologies is shown in Fig 4B. Here, knockdown of Klaroid resulted in large and asymmetric nuclei, while knockdown of Msp300 resulted in smaller rounded nuclei. This contrasted lamin knockdown cells where nuclei were similar in shape to the control nuclei. To obtain an understanding of the relevance of cytoskeleton mediated gene expression changes on cellular functioning we employed a gene ontology (GO)

57

analysis. Illustrated in Fig 4C-E

are pie chart annotations of GO groups enriched in each of the differential gene expressions with respect to control, following introduction of the cytoskeletal perturbations. The GO analysis of Msp300 (Fig 4E) illustrated an up-regulation of gene clusters involved in the regulation of programmed cell death and cell motility including microtubule-based processes and organization; with a down-regulation of gene groups associated with regulation of cell adhesion and cell cycle. This is consistent with our earlier studies which show enhanced nuclear dynamics

ACCEPTED MANUSCRIPT on actin perturbation (Fig 3E), as well as studies from literature which demonstrate that genes that affect nuclear morphology show enhanced cell proliferation

58,59

. In contrast, the GO

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analysis of Klaroid (Fig 4C) revealed an up-regulation of gene clusters involved in the

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organization of actin cytoskeleton and plasma membrane, as well as transcription regulation; and down-regulation of GO groups associated with regulation of apoptosis, cell motility and negative

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regulation of cell cycle. These contrasting gene enrichment groups reveal the differential role of

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perinuclear actin-microtubule mechanical influence on cellular functioning. The gene cluster analysis of Lamin (Fig 4D) revealed similarities with Msp300, reinforcing the functional 60

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influence that the nucleo-cytoskeleton has mechanically on the cell

. Futhermore, the GO

analysis highlights that perturbation to Lamin organization results in a down-regulation of cell

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proliferation and cell cycle gene groups which is consistent with earlier studies linking lamin functionality to cell proliferation and normal cell cycle progression

61

. Misregulation of

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transcription regulation gene groups, and up-regulation of cell migration also find reflection in studies on nuclear lamina disorders

15,62

. The differential alteration of cytoskeleton seen in the

functional gene annotations is further illustrated in Fig 4F, which provides a heatmap representation of genes that were differentially regulated by Klaroid, Msp300 and Lamin, both with respect to control, as well as to each other. Study of three-way gene expression profile revealed corresponding patterns of altered regulation of actin and microtubule proteins (such as actomyosin filament genes Act57B (CG10067), Myo10A (CG43657), and microtubule nucleation gene (CG18109), tubulin structural component genes αTub67C (CG8308) and γTub37C (CG17566)) in Msp300 and Lamin perturbations. And conversely in Klaroid perturbations, differential regulation of actin binding gene MICAL (CG33208), myosin heavy chain Mhc (CG17927) and Syn2 (CG4905), and microtubule associated dynein heavy chain

ACCEPTED MANUSCRIPT Dhc1 and αTub84B (CG1913). Additionally, the expression profile also illustrated patterns of up regulation of microtubule proteins (such as kinesin associated genes Kif19A (CG9913) and

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Klp61F (CG9191) and microtubule structural component γTub37C (CG17566)) in Klaroid

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perturbations, and conversely up regulation of actomyosin genes (myosin heavy chain Mhc (CG17927) and actin filament genes Act79B (CG7478) and Act42A (CG12051)) in Msp300 and

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Lamin perturbations. This differential regulation demonstrated compensatory cytoskeletal gene

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expression changes on alteration of the cytoskeleton force balance, reminiscent of earlier studies which showed an up-regulation of lamina structural genes in lamin B1 defective cells

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. This

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study supports our evidence of differential cytoskeleton modulation of nuclear mechanics and dynamics, and highlights the influence of cytoskeletal architecture on cellular functioning and

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gene expression 64.

Understanding how the structure and function of the cytoskeleton influences nuclear dynamics

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and function is relevant both in light of the understanding that mechanical forces influence genome function 29,65 and the demonstration that impairment of cell structure and organization is correlated with cellular dysfunction

66,67

. Our work leads us to propose that during normal

cellular function, a force-balance exists between the perinuclear actin and microtubule networks, as a means to tune nuclear morphology and dynamics. This subsequently plays a role in the regulation of chromatin organization and gene expression. Despite these advances in understanding how cellular mechanics influence genome function, many questions remain unanswered, particular in regards to the specific mechanisms that underlie this mechanical influence. Acknowledgements

ACCEPTED MANUSCRIPT This study used the core facilities and the Central Imaging and Flow Facility (CIFF) at the National Centre for Biological Sciences (NCBS), Bangalore. The S2 lamin-GFP and mCherry-

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tubulin cells were a kind gift from Ronald D. Vale, Dept. of Cellular and Molecular

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Pharmacology, UCSF, USA. We are grateful to Steven Wolf (MBI) for editing assistance and Chromous Biotechnology, Bangalore for dsRNA preparation. This work was funded in part by

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the Department of Science and Technology (DST) Nanoscience Initiative grant, India and

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Mechanobiology Institute, Singapore.

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Author Contributions

N.R. and G.V.S. conceived and designed the experiments. N.R. performed experiments. N.R.

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Key words

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and G.V.S. interpreted data and wrote the paper.

actin, microtubule, lamin, histones, mechanotransduction

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1. The perinuclear actin and microtubule cytoskeleton differentially modulate nuclear

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morphology. Changes in nuclear architecture following cytoskeletal perturbation or stabilization.

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Phalloidin-stained actin and α-tubulin antibody stained microtubule organization are represented,

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along with the resulting altered DNA, which is visible by Hoechst 33342 staining. (a) Representative images at the basal plane of the cell demonstrate a cytoskeleton mediated

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modulation of nuclear morphology. (b) Images at the central plane reveal alteration in lateral

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microtubule organization associated with changes in nuclear morphology and positioning (c-d) Representative images of immunostained nuclear lamin Dm0 and its 3D rendering illustrate

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deformation in nuclear periphery with altered perinuclear actin-microtubule force balance (e)

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Quantitative assessment of changes in the nuclear projected area (PA) and height with cytoskeletal alteration. (f) Perturbation to actin-microtubule organization alters nuclear shape

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(SI) and position (PI). Scale bar 5µm; n>50. Nuclear shape index (SI) is defined as 4πA/P2

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where A is the projected area, and P is the perimeter of the nucleus. Nuclear position index (PI) is defined as the offset in localization of nuclei centroid measured from the cell centroid, normalized by the projected area (PA) of the cell. Error bars indicate mean ± SE; *p < 0.05; **p < 0.001; ***p < 1 x 10-4. Drosophila S2R+ cells (Drosophila Genomics Resource Center) were cultured in Schneiders medium with 10% FBS at room temperature and ambient CO2. Cells were fixed using 4% PFA for 20 min and stained with 1.0μg/ml Hoechst 33342 for 15 min to label DNA. Immunofluorescence studies were performed using the following reagents: anti-lamin antibody ADL67.10 (Developmental Studies Hybridoma Bank), anti-tubulin antibody AA4.3 (Developmental Studies Hybridoma Bank), TRITC Phalloidin (Sigma Aldrich), DNA dye Hoechst 33342. Pharmacological inhibitors were used as follows: latrunculin A (LatA: Sigma

ACCEPTED MANUSCRIPT Aldrich) 100nM for 30 min; nocodazole (Noc: Sigma Aldrich) 6 µM for 6 hr; paclitaxel (Pac: Sigma Aldrich) 6µM for 4 hr; and paclitaxel + latrunculin A (Pac+LatA); paclitaxel stabilization

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followed by latrunculin A perturbation by the same procedure. Zeiss 700 microscope was used

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for the confocal imaging experiments, using 63x/1.4 NA oil immersion objective; acquiring images (512x512 pixels, 12 bit images, with optimal pinhole sizes). Z-stacks of nuclei with a

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step size of 0.3-0.75 µm were acquired. The 3d rendering of confocal images was done using

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Imaris 7.6.1 (Bitplane).

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Fig. 2. Actin-microtubule control of nuclear shape and size confirmed using RNAi. (a) Representative images of nuclei, taken at 20x magnification, of (a1) control and (a2) Pav

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(CG1258) RNAi knockdown cells stained with Hoechst 33342. Scale bar 30µm. (b) Schematic

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depicting the methodology used in the selection of the upper and lower bounds for phenotypic characteristics. The morphological change of each perturbed population was described by the log

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mean fold ratio (to base 2) of the fraction of nuclei in the upper/lower bounds of the distribution compared to the control distribution. Upper-bound: fraction of distribution above µ + 0.75σ, and lower-bound: fraction of distribution below µ - 0.75σ, normalized by the same fraction in the control population; where µ, σ correspond to mean and standard deviation of the distribution respectively. (c) Representative images, taken at 63x magnification and stained with Hoechst 33342, demonstrating changes in nuclear morphology with perturbation to cellular components. (c1) Control (c2) Ote (CG5581) (c3) Cup (CG11181) (c4) Bocksbeutel (CG9424) (c5) Ft (CG3352) (c6) L(2)gl (CG2671) (c7) Spag4 (CG6589) (c8) Klarsicht (CG17046). Targeting of nuclear envelope proteins (c1-c4) and cell adhesion/plasma membrane proteins (c5-6) resulted in rounded nuclei (small PA) and some alteration in shape (SI). Targeting of cytoskeleton proteins (c7-8) showed both rounded nuclei (small PA) as well as flattened nuclei having anisotropic

ACCEPTED MANUSCRIPT shapes (large SI). Scale bar 10µm. (d) Representative DNA stained Hoechst 33342 images of nuclei in actomyosin perturbed (d1) Act42a (CG12051), (d2) Myo28b1 (CG6976)) cells, and in

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microtubule and motor perturbed (d3) (gTub23c (CG3157), (d4) Klp68d (CG7293)) cells. Scale

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bar 10µm. (e) Heatmaps illustrating changes in nuclear projected area (PA) and shape index (SI), quantitatively assessed by a log mean fold ratio of the fraction of nuclei in the upper and

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lower bounds of the distributions in perturbed cells compared to control. RNAi was carried out

employing the established procedure

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using the dsRNA prepared from the OpenBiosystems v1 Drosophila RNAi library and : 1x105 cells were incubated with 3μg dsRNA in a

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moist environment for 5 days. Imaging of nuclear morphology was captured on a Nikon 2000TE microscope using a 20x, 0.75 NA objective. Images of > 500 cells were processed via custom

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routines written in MATLAB (MathWorks) to provide nuclear morphometric analysis of nuclear projected area (PA) and shape index (SI).

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Fig. 3. Alteration to nuclear and chromatin dynamics by perinuclear actin-microtubule assemblies. (a) Montage depicting microtubule (red) and lamin (green) organization in laminGFP mCherry-tubulin S2 cells. Images were captured along the height of the nucleus from the basal plane, to the central plane. Images reflect cytoskeletal influences on nuclear morphology. (b) Changes in the nuclei anchorage offset from the cell base (n=20) (c) Changes in heterochromatin organization (along basal to apical planes) within nuclei of cells with altered cytoskeleton architecture, visualized by immunostaining of heterochromatin protein HP1 (antiHP1 antibody ab24726 (Abcam)) and displayed by color-coded intensity images. Z-outlines of heterochromatin (marked in green color) and condensed heterochromatin structures (marked in red color). Condensed heterochromatin structures were identified by automated thresholding across z-slices, using custom routines written in MATLAB (MathWorks, USA); employing the

ACCEPTED MANUSCRIPT image thresholding method graythresh(), with a factor enhanced by 3.5 from that employed for heterochromatin structures. (d) Quantitative assessment of variation in heterochromatin

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organization along the nuclear height. Heterochromatin shape index (SI) is defined as 4πA/P2,

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where A is the projected area, and P is the perimeter of the heterochromatin; with its variation measured along the nuclear height. Condensed heterochromatin position index (PI) is defined as

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the offset in localization of dense heterochromatin structures measured from the heterochromatin

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centroid and normalized by the heterochromatin area, with its variation measured along the nuclear height. Scale bar 5µm. n=30. (e) Assessment of nuclear envelope fluctuation with actin

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(Act5c (CG4027; n=11), Vinc (CG3299; n=10), Msp300 (CG42768; n=12)) and microtubule (βtub56d (CG9277; n=12), Khc (CG7765; n=8)) perturbation (f) Quantitative assessment of

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bleach recoveries in H2B-EGFP nuclei following cytoskeleton perturbation. Zeiss LSM 510Meta (63x/1.4 NA oil immersion objective) and Perkin Elmer spinning disk (100x/1.4 NA oil

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immersion objective) microscopes were used in our imaging experiments. Z-stacks of nuclei with a step size of 0.2–1 µm were acquired. Error bars indicate mean ± SE; **p < 0.05; ***p < 1 x 10-3. The structural fluctuation of the nuclear boundary was analyzed from time-lapse images of the EGFP-tagged histone H2B nuclei of Drosophila S2R+ cells; with nuclear boundary detection and fluctuation calculation made using custom-routines written in MATLAB (MathWorks, USA). The fluctuation in the radial distance of the nuclear boundary <δri> for each angle i (where i = 0º, 1º.. 359º) was assessed by computing the change in radial distance at each time point t with respect to its earlier time point (t-1). The mean squared fluctuation was then calculated as [ < (δr)2 > = Σ (δri)2 / N ], summed over all angles N = Σ i. FRAP experiments on cells were captured with identical bleach settings; fluorescence recovery was monitored in ~1µm sized bleach spots on confocal slices passing through the central plane of the H2B-EGFP

ACCEPTED MANUSCRIPT transfected nuclei, and was quantified by the fraction of intensity recovery; (n = 10 for each perturbation). Transfection experiments were performed using Effectene Transfectene reagent

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(Qiagen) and cells were assayed 36 hrs post transfection.

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Fig. 4. Changes in the transcriptome profile following cytoskeleton alteration. (a) Representative

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images of changes in (a1) control nuclear morphology following perturbation to (a2) microtubules (Klaroid), (a3) nuclear lamina (Lamin) and (a4) actin (Msp300). Scale bar 10µm.

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(b) Quantitative assessment of altered nuclear projected area (PA) and shape index (SI) with

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cytoskeleton reorganization, as described earlier by the log mean fold ratio of the fraction of nuclei in the upper and lower bounds of the distributions in perturbed cells compared to control.

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(c-e) Functional annotation of differential up- and down-regulated genes for (c) Klaroid, (d)

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Lamin and (e) Msp300, using a 1.0 fold-change cutoff. The pie charts depict functionally enriched gene ontology (GO) groups for differential gene expression of (c) Klaroid: analysis of

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261 differentially regulated genes lead to 25 up regulated gene clusters and 42 down regulated gene clusters; and (d) Lamin: analysis of 446 differentially regulated genes lead to 37 up regulated clusters and 59 down regulated gene clusters; and (e) Msp300: analysis of 394 differentially regulated genes lead to 31 up regulated clusters and 55 down regulated gene clusters. (f) Heatmap depicting gene expression changes of top-35 differentially regulated cellular components on cytoskeleton perturbation. Three-way differential gene expression selection criteria includes genes with perturbation/control > 1.0-fold change cutoff; but excludes those genes which are similarly up/down regulated among all three perturbations. Microarray experiments were carried out in duplicate. RNAi knockdown was carried out using a previously described protocol, and RNA extraction was done using RNeasy Minikit (Qiagen, Cat#74104). RNA concentration and purity were determined using Nanodrop® ND-1000 spectrophotometer

ACCEPTED MANUSCRIPT (NanoDrop Technologies, Wilmington, DE) and the integrity of RNA was verified on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChip (Agilent Technologies). RNA was labeled

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using Agilent dye Cy3 CTP and hybridized to Drosophila GeneExpression Array 8x15K

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(AMADID:20441). Slides were scanned using Agilent Microarray Scanner G Model G2565BA at 5µ resolution, and data was extracted from images using Feature Extraction software v 9.5 of

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Agilent. Microarray data was analyzed using custom routines written in MATLAB (MathWorks,

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USA). Background subtracted gene expression data within each array were normalized by their respective means. Expression between replicate genes were used to select similarly expressed

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genes based on the mean (µ) and standard deviation (σ) between gene duplicates: | Gene 1(i) – Gene 2(i) | < µ - 0.5σ, where Gene 1(i) and Gene 2(i) are the two replicates of Gene (i) that are

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selected if the difference between their means lie below a threshold of 0.5 of their standard deviation. Mean expression (of control and perturbed) data was calculated from the expression

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data of replicates as: Gene (i) = Σ ( Gene 1(i), Gene 2(i) ). The fold change in differential gene expression was determined by logarithmic ratio of gene expression in perturbed state vs control state. Fold change (i) = Log 2 ( Perturbed state (i) / Control state (i) ). The quantitative measure used to distinguish differentially expressed genes was determined by a fold change > 1.0 cutoff. Functional analysis of differentially regulated genes was performed using the annotation tool, DAVID Bioinformatics Resources 6.7 (NIAID) NIH 70. Pie charts were assembled from the GO enriched functional annotations.

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Graphical abstract

ACCEPTED MANUSCRIPT Highlights 

It is known that the mechanical integrity of the nucleus is influenced by nucleo-cytoskeletal assemblies. However the coupling between microtubule- and actin-driven cytoskeleton

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components in controlling nuclear morphology and chromatin organization are not well understood.

We demonstrate the role of baso-lateral microtubule assemblies and the influence of

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 

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perinuclear actin-microtubule force-balance on nuclear morphology. We show that the perinuclear actin-microtubule cytoskeleton architecture results in alteration

histone mobility. 

We highlight the functional relevance of changes in the transcriptome profile with

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cytoskeleton network alteration.

In summary, we propose that coordinated perinuclear actin-microtubule organization

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influences nuclear spatial architecture and chromatin dynamics.

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

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in nuclear dynamics and lamin integrity, and influences heterochromatin localization and core