Perinuclear Actin Research Articles

DATA DRIVEN AND CELL SPECIFIC DETERMINATION OF NUCLEI-ASSOCIATED ACTIN STRUCTURE

In the specialized field of cellular mechanosignaling, the intricate relationship between the cytoskeleton and nucleus is crucial for determining the behavior and fate of mesenchymal stem cells (MSCs). Existing analytical methods for filamentous actin fibers (F-actin) suffer from issues of accuracy and reproducibility. To address this, our research introduces a significant advancement in image analysis by utilizing two deep-learning segmentation Convolutional Neural Networks (CNNs) based on U-Net architecture. This algorithm precisely quantifies F-actin and offers reproducible data from 3D confocal microscopy images. Our CNNs automatically extract the 3D shape of both the nucleus and individual actin fibers, thereby enabling the geometric reconstruction of these critical cellular components. We applied this methodology to MSCs exposed to low-intensity vibration, and hypothesized that mechanical stimuli will induce perinuclear actin remodeling. Confocal Z-stack images of MSCs were captured using a 40x oil lens and a Zeiss LSM 900 microscope. Nucleus and F-actin structures were stained with Hoechst 33342 and Phalloidin, respectively. Nucleus-based images were employed to mask and isolate actin fibers within the nuclear vicinity. A specialized deep-learning segmentation model was engineered within the PyTorch framework. For model training, a selective 2% of nuclei and F-actin images were annotated. Specifically, three layers from each of six representative nuclei, among approximately 500 cross-sectional layers, were meticulously selected; a similar approach was employed for actin. The model underwent 200 epochs of training. Post-training, the deep-learning model was employed to process all collected cross-sectional images of F-actin and nuclei. A subsequent analytical step involved the scrutiny of intersecting segmented fibers between consecutive layers for geometrically accurate actin fiber reconstruction. We analyzed 102 control and 110 vibration-treated mesenchymal stem cells (MSCs) by utilizing our deep-learning-based reconstruction algorithm on confocal Z-stack images. Our data revealed an 8% decrease in nuclear height from 5.73 µm to 5.28 µm (p < 0.001) upon application of low-intensity vibration. In contrast, the two groups observed no significant difference in nuclear volume. Accompanying these changes at the nuclear level, the following changes in the F-actin cytoskeleton were observed upon LIV application. The total number of actin filaments per cell increased from 117 to 150. The average volume of individual actin fibers rose from 0.40 µm³ to 0.43 µm³. Notably, the volume of fibers at the apical nuclear surface increased from 0.35 µm³ to 0.46 µm³. While the average length of actin fibers in the apical nuclear surface remained largely unchanged due to the addition of smaller fibers in LIV groups, the LIV group had a larger number of fibers that were larger than 15µm.

P300 Modulates Endothelial Mechanotransduction of Fluid Shear Stress.

P300 is a lysine acetyltransferase that plays a significant role in regulating transcription and the nuclear acetylome. While P300 has been shown to be required for the transcription of certain early flow responsive genes, relatively little is known about its role in the endothelial response to hemodynamic fluid stress. Here we sought to define the role of P300 in mechanotransduction of fluid shear stress in the vascular endothelium. To characterize cellular mechanotransduction and physical properties after perturbation of P300, we performed bulk RNA sequencing, confocal and Brillouin microscopy, and functional assays on HUVEC. Inhibition of P300 in HUVEC triggers a hyper-alignment phenotype, with cells aligning to flow sooner and more uniformly in the presence of the P300 inhibitor A-485 compared to load controls. Bulk transcriptomics revealed differential expression of genes related to the actin cytoskeleton and migration in cells exposed to A-485. Scratch wound and bead sprouting assays demonstrated that treatment with A-485 increased 2D and 3D migration of HUVEC. Closer examination of filamentous actin revealed the presence of a perinuclear actin cap in both P300 knockdown HUVEC and HUVEC treated with A-485. Interrogation of cell mechanical properties via Brillouin microscopy demonstrated that HUVEC treated with A-485 had lower Brillouin shifts in both the cell body and the nucleus, suggesting that P300 inhibition triggers an increase in cellular and nuclear compliance. Together, these results point to a novel role of P300 in modulating endothelial cell mechanics and mechanotransduction of hemodynamic shear stress. The online version contains supplementary material available at 10.1007/s12195-024-00805-2.

Coupling of Perinuclear Actin Cap and Nuclear Mechanics in Regulating Flow-Induced Yap Spatiotemporal Nucleocytoplasmic Transport.

Mechanical forces, including flow shear stress, govern fundamental cellular processes by modulating nucleocytoplasmic transport of transcription factors like Yes-associated Protein (YAP). However, the underlying mechanical mechanism remains elusive. In this study, it is reported that unidirectional flow induces biphasic YAP transport with initial nuclear import, followed by nuclear export as actin cap formation and nuclear stiffening. Conversely, pathological oscillatory flow induces slight actin cap formation, nuclear softening, and sustained YAP nuclear localization. To elucidate the disparately YAP spatiotemporal distribution, a 3D mechanochemical model is developed, which integrates flow sensing, cytoskeleton organization, nucleus mechanotransduction, and YAP transport. The results unveiled that despite the significant localized nuclear stress imposed by the actin cap, its inherent stiffness counteracts the dispersed contractile stress exerted by conventional fibers on the nuclear membrane. Moreover, alterations in nuclear stiffness synergistically regulate nuclear deformation, thereby governing YAP transport. Furthermore, by expanding the single-cell model to a collective vertex framework, it is revealed that the irregularities in actin cap formation within individual cells have the potential to induce topological defects and spatially heterogeneous YAP distribution in the cellular monolayer. This work unveils a unified mechanism of flow-induced nucleocytoplasmic transport, providing a linkage between transcription factor localization and mechanical stimulation.

Aberrant MET activation impairs perinuclear actin cap organization with YAP1 cytosolic relocation

Little is known about the signaling network responsible for the organization of the perinuclear actin cap, a recently identified structure holding unique roles in the regulation of nuclear shape and cell directionality. In cancer cells expressing a constitutively active MET, we show a rearrangement of the actin cap filaments, which crash into perinuclear patches associated with spherical nuclei, meandering cell motility and inactivation of the mechano-transducer YAP1. MET ablation is sufficient to reactivate YAP1 and restore the cap, leading to enhanced directionality and flattened nuclei. Consistently, the introduction of a hyperactive MET in normal epithelial cells, enhances nuclear height and alters the cap organization, as also confirmed by TEM analysis. Finally, the constitutively active YAP1 mutant YAP5SA is able to overcome the effects of oncogenic MET. Overall, our work describes a signaling axis empowering MET-mediated YAP1 dampening and actin cap misalignment, with implications for nuclear shape and cell motility.

FHODs: Nuclear tethered formins for nuclear mechanotransduction.

In this review, we discuss FHOD formins with a focus on recent studies that reveal a new role for them as critical links for nuclear mechanotransduction. The FHOD family in vertebrates comprises two structurally related proteins, FHOD1 and FHOD3. Their similar biochemical properties suggest overlapping and redundant functions. FHOD1 is widely expressed, FHOD3 less so, with highest expression in skeletal (FHOD1) and cardiac (FHOD3) muscle where specific splice isoforms are expressed. Unlike other formins, FHODs have strong F-actin bundling activity and relatively weak actin polymerization activity. These activities are regulated by phosphorylation by ROCK and Src kinases; bundling is additionally regulated by ERK1/2 kinases. FHODs are unique among formins in their association with the nuclear envelope through direct, high affinity binding to the outer nuclear membrane proteins nesprin-1G and nesprin-2G. Recent crystallographic structures reveal an interaction between a conserved motif in one of the spectrin repeats (SRs) of nesprin-1G/2G and a site adjacent to the regulatory domain in the amino terminus of FHODs. Nesprins are components of the LINC (linker of nucleoskeleton and cytoskeleton) complex that spans both nuclear membranes and mediates bidirectional transmission of mechanical forces between the nucleus and the cytoskeleton. FHODs interact near the actin-binding calponin homology (CH) domains of nesprin-1G/2G enabling a branched connection to actin filaments that presumably strengthens the interaction. At the cellular level, the tethering of FHODs to the outer nuclear membrane mechanically couples perinuclear actin arrays to the nucleus to move and position it in fibroblasts, cardiomyocytes, and potentially other cells. FHODs also function in adhesion maturation during cell migration and in the generation of sarcomeres, activities distant from the nucleus but that are still influenced by it. Human genetic studies have identified multiple FHOD3 variants linked to dilated and hypertrophic cardiomyopathies, with many mutations mapping to "hot spots" in FHOD3 domains. We discuss how FHOD1/3's role in reinforcing the LINC complex and connecting to perinuclear actin contributes to functions of mechanically active tissues such as striated muscle.

Tissue fluidification promotes a cGAS–STING cytosolic DNA response in invasive breast cancer

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS–STING (cyclic GMP-AMP synthase–signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.

Programmable integrin and N-cadherin adhesive interactions modulate mechanosensing of mesenchymal stem cells by cofilin phosphorylation

During mesenchymal development, the sources of mechanical forces transduced by cells transition over time from predominantly cell-cell interactions to predominantly cell-extracellular matrix (ECM) interactions. Transduction of the associated mechanical signals is critical for development, but how these signals converge to regulate human mesenchymal stem cells (hMSCs) mechanosensing is not fully understood, in part because time-evolving mechanical signals cannot readily be presented in vitro. Here, we established a DNA-driven cell culture platform that could be programmed to present the RGD peptide from fibronectin, mimicking cell-ECM interactions, and the HAVDI peptide from N-cadherin, mimicking cell-cell interactions, through DNA hybridization and toehold-mediated strand displacement reactions. The platform could be programmed to mimic the evolving cell-ECM and cell-cell interactions during mesenchymal development. We applied this platform to reveal that RGD/integrin ligation promoted cofilin phosphorylation, while HAVDI/N-cadherin ligation inhibited cofilin phosphorylation. Cofilin phosphorylation upregulated perinuclear apical actin fibers, which deformed the nucleus and thereby induced YAP nuclear localization in hMSCs, resulting in subsequent osteogenic differentiation. Our programmable culture platform is broadly applicable to the study of dynamic, integrated mechanobiological signals in development, healing, and tissue engineering.

Neospora caninum Infection Triggers S-phase Arrest and Alters Nuclear Characteristics in Primary Bovine Endothelial Host Cells.

Neospora caninum represents a major cause of abortive disease in bovines and small ruminants worldwide. As a typical obligate intracellular apicomplexan parasite, N. caninum needs to modulate its host cell for successful replication. In the current study, we focused on parasite-driven interference with host cell cycle progression. By performing DNA content-based cell cycle phase analyses in N. caninum-infected primary bovine umbilical vein endothelial cells (BUVEC), a parasite-driven S-phase arrest was detected at both 24 and 32 h p. i., being paralleled by fewer host cells experiencing the G0/G1 cell cycle phase. When analyzing S-subphases, proliferation cell nuclear antigen (per PCNA)-based experiments showed a reduced population of BUVEC in the late S-phase. Analyses on key molecules of cell cycle regulation documented a significant alteration of cyclin A2 and cyclin B1 abundance in N. caninum-infected host endothelial cells, thereby confirming irregularities in the S-phase and S-to-G2/M-phase transition. In line with cell cycle alterations, general nuclear parameters revealed smaller nuclear sizes and morphological abnormalities of BUVEC nuclei within the N. caninum-infected host cell layer. The latter observations were also confirmed by transmission electron microscopy (TEM) and by analyses of lamin B1 as a marker of nuclear lamina, which illustrated an inhomogeneous nuclear lamin B1 distribution, nuclear foldings, and invaginations, thereby reflecting nuclear misshaping. Interestingly, the latter finding applied to both non-infected and infected host cells within parasitized BUVEC layer. Additionally, actin detection indicated alterations in the perinuclear actin cap formation since typical nucleo-transversal filaments were consistently lacking in N. caninum-infected BUVEC, as also documented by significantly decreased actin-related intensities in the perinuclear region. These data indicate that N. caninum indeed alters host cell cycle progression and severely affects the host cell nuclear phenotype in primary bovine endothelial host cells. In summary, these findings add novel data on the complex N. caninum-specific modulation of host cell and nucleus, thereby demonstrating clear differences in cell cycle progression modulation driven by other closely related apicomplexans like Toxoplasma gondii and Besnotia besnoiti.

YAP/TAZ activity in stromal cells prevents ageing by controlling cGAS-STING.

Ageing is intimately connected tothe induction of cell senescence1,2, but why this is so remains poorly understood. A key challenge is the identification of pathways thatnormally suppress senescence, arelost during ageing and arefunctionally relevant to oppose ageing3. Here we connected the structural and functional decline of ageing tissues to attenuated function of themaster effectors of cellular mechanosignallingYAP and TAZ. YAP/TAZ activity declines during physiological ageing in stromal cells, and mimicking such decline through genetic inactivation ofYAP/TAZin these cellsleads to accelerated ageing. Conversely, sustaining YAP function rejuvenates old cells and opposes the emergence of ageing-related traits associated with either physiological ageing or accelerated ageing triggered by a mechano-defective extracellular matrix. Ageing traits induced by inactivation ofYAP/TAZ are preceded by induction of tissue senescence. This occurs because YAP/TAZ mechanotransduction suppresses cGAS-STING signalling, to the extent thatinhibition of STING prevents tissue senescence and premature ageing-related tissue degeneration after YAP/TAZ inactivation. Mechanistically, YAP/TAZ-mediated control of cGAS-STING signallingrelies on theunexpected roleof YAP/TAZ in preserving nuclear envelope integrity, at least in part through direct transcriptional regulation of lamin B1 and ACTR2, the latter of which isinvolved in building the peri-nuclear actin cap. The findings demonstrate that decliningYAP/TAZ mechanotransduction drives ageing by unleashing cGAS-STINGsignalling, a pillar of innate immunity. Thus, sustaining YAP/TAZ mechanosignalling or inhibiting STING may represent promising approaches for limitingsenescence-associated inflammation and improving healthy ageing.

Gravity-Vector Induces Mechanical Remodeling of rMSCs via Combined Substrate Stiffness and Orientation.

Distinct physical factors originating from the cellular microenvironment are crucial to the biological homeostasis of stem cells. While substrate stiffness and orientation are known to regulate the mechanical remodeling and fate decision of mesenchymal stem cells (MSCs) separately, it remains unclear how the two factors are combined to manipulate their mechanical stability under gravity vector. Here we quantified these combined effects by placing rat MSCs onto stiffness-varied poly-dimethylsiloxane (PDMS) substrates in upward (180°), downward (0°), or edge-on (90°) orientation. Compared with those values onto glass coverslip, the nuclear longitudinal translocation, due to the density difference between the nucleus and the cytosol, was found to be lower at 0° for 24 h and higher at 90° for 24 and 72 h onto 2.5 MPa PDMS substrate. At 0°, the cell was mechanically supported by remarkably reduced actin and dramatically enhanced vimentin expression. At 90°, both enhanced actin and vimentin expression worked cooperatively to maintain cell stability. Specifically, perinuclear actin stress fibers with a large number, low anisotropy, and visible perinuclear vimentin cords were formed onto 2.5 MPa PDMS at 90° for 72 h, supporting the orientation difference in nuclear translocation and global cytoskeleton expression. This orientation dependence tended to disappear onto softer PDMS, presenting distinctive features in nuclear translocation and cytoskeletal structures. Moreover, cellular morphology and focal adhesion were mainly affected by substrate stiffness, yielding a time course of increased spreading area at 24 h but decreased area at 72 h with a decrease of stiffness. Mechanistically, the cell tended to be stabilized onto these PDMS substrates via β1 integrin–focal adhesion complexes–actin mechanosensitive axis. These results provided an insight in understanding the combination of substrate stiffness and orientation in defining the mechanical stability of rMSCs.

LINC complex independent perinuclear actin organization and cell migration

The link of the metazoan nucleus to the actin cytoskeleton is highly important for actin polymerization and migration of multiple cell types as well as for mechanotransduction and even affects the cellular transcriptome. Several mechanisms of organization of actin filaments next to the nuclear envelope have been identified. Among these mechanisms the most studied one is the Linker of nucleoskeleton and cytoskeleton (LINC) complex-dependent perinuclear actin organization. However, recently additional mechanisms have been identified: an Actin-related protein-2/3 (Arp2/3)-dependent perinuclear actin polymerization during migration of dendritic cells and a perinuclear actin rim that is formed in response to external force application or migration cues. In parallel, there are also reports on cancer cells that migrate in a LINC complex independent manner and on cancers with reduced expression of the LINC complex components. Thus, suggesting that LINC complex independent migration may be associated with tumour formation.

Palmdelphin Regulates Nuclear Resilience to Mechanical Stress in the Endothelium.

PALMD (palmdelphin) belongs to the family of paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis and predict severity of the disease. Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, coimmunoprecipitation, and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in small interferring RNA-treated EC cultures, knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the single nucleotide polymorphism rs7543130 informed about gene regulatory changes. ECs express the cytosolic PALMD-KKVI splice variant, which associated with RANGAP1 (RAN GTP hydrolyase activating protein 1). RANGAP1 regulates the activity of the GTPase RAN and thereby nucleocytoplasmic shuttling via XPO1 (Exportin1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently in gene regulation because of the effect on localization of transcriptional regulators. Changes in EC responsiveness on loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd-/- mouse aorta, and human aortic valve samples derived from patients with calcific aortic valve stenosis. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD's contribution to calcific aortic valve stenosis pathology.

The actin networks of chytrid fungi reveal evolutionary loss of cytoskeletal complexity in the fungal kingdom

Cells from across the eukaryotic tree use actin polymer networks for a wide variety of functions, including endocytosis, cytokinesis, and cell migration. Despite this functional conservation, the actin cytoskeleton has undergone significant diversification, highlighted by the differences in the actin networks of mammalian cells and yeast. Chytrid fungi diverged before the emergence of the Dikarya (multicellular fungi and yeast) and therefore provide a unique opportunity to study actin cytoskeletal evolution. Chytrids have two life stages: zoospore cells that can swim with a flagellum and sessile sporangial cells that, like multicellular fungi, are encased in a chitinous cell wall. Here, we show that zoospores of the amphibian-killing chytrid Batrachochytrium dendrobatidis (Bd) build dynamic actin structures resembling those of animal cells, including an actin cortex, pseudopods, and filopodia-like spikes. In contrast, Bd sporangia assemble perinuclear actin shells and actin patches similar to those of yeast. The use of specific small-molecule inhibitors indicate that nearly all of Bd's actin structures are dynamic and use distinct nucleators: although pseudopods and actin patches are Arp2/3 dependent, the actin cortex appears formin dependent and actin spikes require both nucleators. Our analysis of multiple chytrid genomes reveals actin regulators and myosin motors found in animals, but not dikaryotic fungi, as well as fungal-specific components. The presence of animal- and yeast-like actin cytoskeletal components in the genome combined with the intermediate actin phenotypes in Bd suggests that the simplicity of the yeast cytoskeleton may be due to evolutionary loss.

Increased Lamin B1 Levels Promote Cell Migration by Altering Perinuclear Actin Organization.

Cell migration requires reposition and reshaping of the cell nucleus. The nuclear lamina is highly important for migration of both primary and cancer cells. B-type lamins are important for proper migration of epicardial cells and neurons and increased lamin B to lamin A ratio accelerates cancer cell migration through confined spaces. Moreover, a positive association between lamin B1 levels and tumor formation and progression is found in various cancer types. Still, the molecular mechanism by which B-type lamins promote cell migration is not fully understood. To better understand this mechanism, we tested the effects of lamin B1 on perinuclear actin organization. Here we show that induction of melanoma cell migration leads to the formation of a cytosolic Linker of Nucleoskeleton and Cytoskeleton (LINC) complex-independent perinuclear actin rim, which has not been detected in migrating cells, yet. Significantly, increasing the levels of lamin B1 but not the levels of lamin A prevented perinuclear actin rim formation while accelerated the cellular migration rate. To interfere with the perinuclear actin rim, we generated a chimeric protein that is localized to the outer nuclear membrane and cleaves perinuclear actin filaments in a specific manner without disrupting other cytosolic actin filaments. Using this tool, we found that disruption of the perinuclear actin rim accelerated the cellular migration rate in a similar manner to lamin B1 over-expression. Taken together, our results suggest that increased lamin B1 levels can accelerate cell migration by inhibiting the association of the nuclear envelope with actin filaments that may reduce nuclear movement and deformability.

Harnessing the perinuclear actin cap (pnAC) to influence nanocarrier trafficking and gene transfection efficiency in skeletal myoblasts using nanopillars

Gene transfection is important in biotechnology and is used to modify cells intrinsically. It can be conducted in cell suspension or after cell adhesion, where the efficiency is dependent on many factors such as the type of nanocarrier used and cell division processes. Anchor-dependent cells are sensitive to the substrate they are attached to and adapt their behavior accordingly, including plasmid trafficking during gene transfection. Previously, it was shown in our group that the cytoskeleton is an essential factor in influencing gene transfection in skeletal myoblasts using nanogrooves as a substrate. In this study, the effect of the cytoskeleton on gene transfection efficiency of skeletal myoblasts was studied using various nanopillars and nanocarriers. Nanopillars with different diameters (200-1000 nm) and depths (200 or 400 nm) were fabricated using colloidal self-assembly and reactive ion etching. All surfaces were treated with oxygen plasma or polydopamine (PD) to further control cell morphology. Plasmid DNA was delivered into cells using jetPRIME or Lipofectamine 3000 nanocarriers. After screening hundreds of images, two distinguishable F-actin distributions were found, i.e., cells with or without a perinuclear actin cap (pnAC). Cells attached to nanopillars, especially the deep pillars, had a smaller spreading area, shorter F-actin, more 3D-like cell nuclei, and a lower percentage of pnAC, which lead to a higher gene transfection efficiency using jetPRIME. On the other hand, cells attached to the shallow nanopillars or flat surfaces had a larger spreading area, longer F-actin, more 2D-like cell nuclei, and a higher percentage of pnAC that facilitates gene transfection using Lipofectamine. The effects of cell density, cytoskeleton (cytoD), and focal adhesions (RGD) on gene transfection were also studied, and the results were consistent with our hypothesis that F-actin distribution is one of the critical factors in gene transfection. In conclusion, pnAC plays a vital role in the intracellular trafficking of nanocarrier/plasmid complexes and this study provides new insights into gene transfection in anchor-dependent cells. Statement of SignificanceThis study provides a new perspective in gene transfection using attached cells where perinuclear actin cap (pnAC) is an essential factor involved in transfection efficiency. A series of nanopillars were used to harness cell and cytoskeleton morphology. Two distinguishable cytoskeletal structures were found including cells with or without pnAC. 2D-like cells with pnAC facilitate gene delivery using liposome-based nanocarriers, while 3D-like cells without pnAC benefit gene delivery using cationic polymer-based nanocarriers. This study reveals the importance of the cytoskeleton during gene transfection that is beneficial in tissue transfection.

Prophase-Specific Perinuclear Actin Coordinates Centrosome Separation and Positioning to Ensure Accurate Chromosome Segregation

SummaryCentrosome separation in late G2/ early prophase requires precise spatial coordination that is determined by a balance of forces promoting and antagonizing separation. The major effector of centrosome separation is the kinesin Eg5. However, the identity and regulation of Eg5-antagonizing forces is less well characterized. By manipulating candidate components, we find that centrosome separation is reversible and that separated centrosomes congress toward a central position underneath the flat nucleus. This positioning mechanism requires microtubule polymerization, as well as actin polymerization. We identify perinuclear actin structures that form in late G2/early prophase and interact with microtubules emanating from the centrosomes. Disrupting these structures by breaking the interactions of the linker of nucleoskeleton and cytoskeleton (LINC) complex with perinuclear actin filaments abrogates this centrosome positioning mechanism and causes an increase in subsequent chromosome segregation errors. Our results demonstrate how geometrical cues from the cell nucleus coordinate the orientation of the emanating spindle poles before nuclear envelope breakdown.

Targeting Tumor Cells by Actin-Regulated Nuclear Envelope Rupture

The survival and plasticity of cancer cells depend of numerous factors which distinguish them from normal cells. Epithelial cancers characteristically deregulate cell mechanics. Tumor cells often show reduced stiffness and generate more contractile forces, as a result of both cellular intrinsic oncogenic alterations and microenvironment changes. Notably, changes in nuclear envelope protein expression, that modify nuclear shape and rigidity, influence cell invasiveness. However, a decrease in nuclear stiffness can lead to nuclear envelope rupture due to focal forces generated by the perinuclear actin structures in metastazing cells that migrate through tight interstitial spaces. Therefore, it has been suggested that an increase propensity to nuclear rupturing represents a new cancer cell vulnerability, which could promote genomic instability. Viral proteins with a tumor cell-selective killing activity offer powerful tools to uncover fundamental differences in cancer cells. In this study, we sought to interrogate the relevance of actomyosin contractility for the tumor cell-selective action of the adenovirus (Ad) type 2 early region 4 (E4) ORF4 (E4orf4) along with the mechanisms involved. We found that E4orf4 tumoricidal activity correlates with actomyosin-driven changes in nuclear shape. Live cell imaging revealed that E4orf4 promoted repeated cycles of nuclear envelope rupture and repair in various tumor cell lines, which caused the nuclear efflux of E4orf4 and exacerbated actomyosin contractility. Such process ultimately led to a prolonged loss of nuclear compartment integrity, overcoming the cellular capacity to repair the nuclear envelope. Furthermore, E4orf4-induced nuclear rupture, as other death-associated endpoint phenotypes, were impaired by depletion of LINC complex proteins. These results suggest that E4orf4 toxicity is regulated nucleo-cytoskeletal connections. We propose that E4orf4 harnesses nuclear envelope mechanical regulation to kill tumor cells, hence offering a unique tool to decipher novel pathways controlling cell invasiveness.

Methods to Measure Perinuclear Actin Dynamics During Nuclear Movement in Migrating Cells.

The nucleus is specifically positioned within a cell in diverse biological contexts. There are multiple connections between the nuclear envelope and the cytoskeleton and these connections are involved in nuclear positioning. During cell polarization prior to cell migration, nuclear envelope proteins bind to the actin cytoskeleton and get organized into linear arrays, known as transmembrane actin-associated nuclear (TAN) lines to move the nucleus away from the leading edge. Here we describe methods to study perinuclear actin dynamics, including measurement of the thickness of actin cables coupled to TAN lines, measurement of the number of perinuclear actin cables, and ablation of perinuclear actin cables. These methods are used to identify mechanisms of nuclear positioning.

The migration direction of hair cell nuclei is closely related to the perinuclear actin filaments in Arabidopsis

Nuclear migration in Arabidopsis root hairs is bidirectional and relies on actin filaments. However, how actin filaments regulate the bidirectional movement of nuclei remains unclear. Here, we discovered that nuclei migrate forward and backward according to the developmental stage of the hair cells. In addition, the migration direction of nuclei was not constant but reversed occasionally, accompanied by nuclear shape changes. Confocal microscopic analysis revealed that perinuclear actin bundles were closely related to the migration and shape of hair cell nuclei. Pharmacological studies showed that SMIFH2, an inhibitor of the actin nucleator-formin, inhibited nuclear backward migration probably by impairing the perinuclear actin filaments. These data indicate that nuclear migration in hair cells is likely motivated by the competition of mechanical forces acting on the nucleus. Furthermore, the perinuclear actin filaments are closely related to the migration direction of hair cell nuclei.

Anisotropy vs isotropy in living cell indentation with AFM

The measurement of local mechanical properties of living cells by nano/micro indentation relies on the foundational assumption of locally isotropic cellular deformation. As a consequence of assumed isotropy, the cell membrane and underlying cytoskeleton are expected to locally deform axisymmetrically when indented by a spherical tip. Here, we directly observe the local geometry of deformation of membrane and cytoskeleton of different living adherent cells during nanoindentation with the integrated Atomic Force (AFM) and spinning disk confocal (SDC) microscope. We show that the presence of the perinuclear actin cap (apical stress fibers), such as those encountered in cells subject to physiological forces, causes a strongly non-axisymmetric membrane deformation during indentation reflecting local mechanical anisotropy. In contrast, axisymmetric membrane deformation reflecting mechanical isotropy was found in cells without actin cap: cancerous cells MDA-MB-231, which naturally lack the actin cap, and NIH 3T3 cells in which the actin cap is disrupted by latrunculin A. Careful studies were undertaken to quantify the effect of the live cell fluorescent stains on the measured mechanical properties. Using finite element computations and the numerical analysis, we explored the capability of one of the simplest anisotropic models – transverse isotropy model with three local mechanical parameters (longitudinal and transverse modulus and planar shear modulus) – to capture the observed non-axisymmetric deformation. These results help identifying which cell types are likely to exhibit non-isotropic properties, how to measure and quantify cellular deformation during AFM indentation using live cell stains and SDC, and suggest modelling guidelines to recover quantitative estimates of the mechanical properties of living cells.