Cytoskeleton

The actomyosin cytoskeleton plays important roles in cell-cell adhesion by generating and responding to forces. Actin-binding proteins support actomyosin networks by reinforcing actin filaments, promoting actin remodeling, and transmitting forces to transmembrane adhesion proteins. One family of actin-binding proteins, LIM domain-containing proteins, is recruited to strained actin filaments. Here, we investigate two members of the LIM domain family, Zyxin and Lipoma-Preferred Partner (LPP), using the embryonic epithelium of Xenopus laevis, where actin-associated cell-cell junctions connect cells to promote tissue integrity and barrier function. Specifically, we compare Zyxin and LPP's response to increased tension at tricellular junctions (TCJs), sites of heightened mechanical strain within the tissue. Upon increased tension, Zyxin and LPP mechanoaccumulate to different extents relative to F-actin, suggesting distinct mechanisms. Our results demonstrate that Zyxin's and LPP's LIM domain-containing regions (LCRs) are sufficient for mechanoaccumulation as well as responsible for the difference in mechanoaccumulation, while their N-termini play a regulatory role in their mechanosensitive responses. Docking simulations show that a hydrophobic interaction between the LCR and the "crack site" that forms in F-actin filaments under high force may underlie the LCR's sensing of strained F-actin. Additionally, the docking simulations reveal that previously-identified conserved residues are present at the binding interface. Mutations of conserved residues in Zyxin or LPP reduce the extent of mechanoaccumulation at TCJs, supporting the docking prediction. Together, this study advances our understanding of Zyxin and LPP's sensing of strained actin at TCJs under mechanical challenge.

Microtubules (MTs) undergo continuous cycles of growth and disassembly. Because the transitions between these states are stochastic, MT age varies widely within a population. As MTs age, they are thought to accumulate post-translational modifications (PTMs) that, directly or indirectly, enhance their stability and thereby extend their lifetime. The rare MTs that withstand prolonged exposure to destabilizing drugs such as nocodazole (NZ) are indeed enriched in PTMs; yet the relationships between MT age, PTMs and stability remain unclear. Using microinjection of labelled tubulin, we measured microtubule network turnover in immortalized mouse embryonic fibroblasts. Half of the network was renewed within 3 minutes and 80% within 10 minutes, while approximately 5% of microtubules persisted for more than 20 minutes. These dynamics were comparable in quiescent and senescent cells, although the fraction of slowly renewing or non-renewing microtubules rose to 20% in senescent cells. Unexpectedly, neither the amount of PTMs (acetylation and detyrosination) nor resistance to NZ increased with MT age, and resistance to NZ was independent of these PTMs. Degrees of acetylation and detyrosination should therefore not be taken as readouts of MT age or stability. Because these PTMs do not accumulate on MTs over time, the chemical modification of polymerized tubulin is likely more reversible and dynamic than previously assumed.

Tropomyosins play a central role in the regulation of the actin cytoskeleton by assembling along filaments into rope-like, head-to-tail polymers ("clusters") along filaments that regulate the binding and activity of numerous actin-binding proteins. Importantly, in cells, tropomyosins do not decorate actin filaments uniformly; some filaments are devoid of tropomyosins, whereas others recruit specific isoforms. To explore the diversity of tropomyosin-isoform binding kinetics and identify factors that control their assembly, we designed tropomyosin constructs, bearing a natural N-terminal acetylation and targeted cysteine mutations for fluorescence labelling. We analysed the binding of different tropomyosin isoforms to individual actin filaments reconstituted inside a microfluidic chamber. We show that, despite significant differences in spontaneous nucleation and elongation rates, all tested tropomyosin isoforms can coassemble within the same clusters. In addition, we identified tropomodulin and leiomodin, two pointed-end-binding proteins, to efficiently nucleate the formation of tropomyosin clusters on actin filaments, for all tested tropomyosin isoforms. Notably, while tropomodulin nucleates tropomyosin clusters from the pointed end, leiomodin does so along the entire filament.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

Centrosome positioning is governed by mechanical interactions between the actin and microtubule cytoskeletons, yet the contribution of different actin architectures and their attachment to the cell membrane remains unclear. We use numerical simulations to study a centrosomal array of microtubules interacting with cortical, bulk, and centrosome-associated actin networks, including passive cross-linkers, motor proteins, and ERM-mediated coupling of actin to the cell membrane. We find that bulk and centrosome-associated actin alone are insufficient for robust centrosome centering, whereas sufficiently dense cortical actin promotes centering through steric interactions with growing microtubules. ERM-mediated attachment of cortical actin to the cell membrane enhances this effect, allowing stable centering at lower actin densities. In a two-centrosome system, microtubule interactions generate a spindle-like structure, while the organization of actin and its membrane attachment through ERM regulate centrosome separation. Antagonistic microtubule motors further limit centrosome separation and establish a finite spindle length. These results demonstrate that ERM-mediated coupling of actin to the cell membrane provides an important mechanical link between the actin cortex and microtubule-driven centrosome positioning, influencing both centrosome centering and spindle geometry.

The spatial organization of chromosomes is crucial for gene regulation and genome stability. Metaphase chromosomes appear physically discrete from one another in conventional chromosome preparations. However, interchromosomal connections, or "linkers", have been observed among mitotic chromosomes, and may play important roles in coordinated chromosome movements and genome stability. We use micropipette-based isolation and manipulation to analyze interchromosome linkers between mammalian mitotic chromosomes. Pulling an isolated chromosome reveals that thin linkers connect them; CREST staining indicates their locations to be at centromeres. The linkers display linear elasticity and have a length-doubling force of approximately 300 pN, comparable to the length-doubling force of an entire metaphase chromosome. Enzymatic treatments demonstrate that the linkers are disrupted by DNase, but not by RNase, protease, or microtubule polymerization (spindle fiber) inhibitors, indicating that linker connectivity is based on DNA. Immunofluorescence experiments indicate that histones and topo II are on the linkers, with topo II organized into discrete foci spaced by approximately 0.4 Mbp. After prolonged metaphase arrest using spindle inhibitors isolated genomes do not display interchromosome linkers. Finally, experiments on intact cells show that interchromosome linkers containing CENP-B can be observed between mitotic chromosomes, indicating they are not an artifact of genome isolation.

One of the first genes identified to regulate the spacing of nuclei in the multinucleated myofiber was Kinesin-1. However, the mechanism by which Kinesin-1 or other kinesins regulate myonuclear spacing is not known. Critically, the myofiber lacks centrosomes, and the many myonuclei act as the primary microtubule organizing centers of the cell. Because of this unique re-structuring, we hypothesized that the kinesins that drive centrosomes apart during mitotic spindle elongation may play a similar role in spacing myonuclei. We found that the bipolar Kinesin-5 (Klp61f) and the (-)-end directed Kinesin-14 (ncd) were both necessary for myonuclear spacing at different times, with both being necessary during embryogenesis, but only ncd being necessary in the fully differentiated myofiber. To investigate the shared mechanisms during embryogenesis, we used live-imaging and found that, similar to the mitotic spindle, Klp61f acts as an accelerator for myonuclear movement, whereas ncd acts as a brake contrary to this movement. To investigate these mechanisms and test the hypothesis that this is dependent on microtubule-sliding, we used super-resolution microscopy to visualize and quantify the microtubule network in animals with disrupted Klp61f or ncd. We found that in both cases, there was a decrease in the amount of microtubule overlap between neighboring myonuclei. Furthermore, we found that disrupting ncd led to severe changes in microtubule network organization, supporting our hypotheses that microtubule-sliding is necessary to space myonuclei, and that ncd likely functions through a unique mechanism in the differentiated myofiber to maintain myonuclear spacing. Together, our data supports a model where myonuclear spacing is regulated by a counteracting force generated by different kinesins during embryonic development. Furthermore, one kinesin, ncd, is repurposed in the differentiated myofiber to dynamically crosslink microtubules, a function necessary to anchor nuclei in place. Thus, kinesin motors regulate myonuclear spacing across developmental time by leveraging opposing forces through diverse mechanisms.

The mechanical properties of the nucleus are critical for maintaining nuclear integrity and function. We previously showed that chromatin dominates short-extension mechanics whereas lamins provide long-extension strain stiffening. To distinguish the roles of lamin isoforms, micromanipulation nucleus force measurements were performed on isolated nuclei from lamin A/C (Lmna-/-) and lamin B1 (Lmnb1-/-) knockout mouse embryonic fibroblast cells. Lamin A/C knockout does not alter short-extension nuclear stiffness but is essential for strain stiffening at longer extensions. Oppositely, lamin B1 loss reduced short-extension stiffness due to facultative heterochromatin loss while long-extension strain stiffening was slightly increased. Loss of lamin A/C and B1 resulted in similar lamin-chromatin linkers effects as LBR did not change and LAP2{beta} decreased in both. A simulation model of a polymeric lamin shell with stiff lamin A/C and softer lamin B1 subunits can qualitatively recapitulate experimental measurements of lamin knockout cells. Lamin A/C knockout resulted in abnormal nuclear shape but not nuclear blebbing or rupture whereas lamin B1 knockout, similar to other perturbations that cause heterochromatin loss, resulted in increased nuclear blebbing and rupture. This work illuminates the distinct mechanical roles of lamin A/C and B1 in determining nuclear structure and integrity.

In epithelial wounds, physically enlarged cells emerge along injury edges where they interact with regular-size cells during collective migration to repair tissue continuity. Although such large cells are often interpreted as migration leaders, recent observations suggest that regular-size cells can reciprocally influence them and that mixed-size cell clusters engage in distinct rotational motion before merging into confluent sheets. How cell size, polarity and adhesive coupling jointly control distinct collective migration modes remains unclear. Because many features of collective epithelial migration, including large and regular-size cell phenotypes, are conserved between in vivo and in vitro systems, here we developed a multi-scale computational model of interacting epithelial cells in two-dimensional culture with dynamic cell-substrate adhesion, cell-cell interactions, protrusion-based polarity, and contact-induced myosin redistribution as polarity regulator. We also explicitly modeled two functionally distinct cell states, featuring regular and large sizes respectively. Simulations showed that symmetric and asymmetric myosin redistribution at cell-cell junctions can produce stable cell contacts and persistent rotational motions by cell doublets. Intriguingly, in mixed-size cell clusters, straight translation can arise both from leader-like large cell and regular-size cell-mediated motion, whereas rotation emerges when large-cell-generated torque overcomes translation while cell-cell adhesion is maintained. Thus, collective migration mode depends on the balance among myosin-driven torque, regular-size cell-mediated translation, substrate coupling, and cell-cell adhesion. These modeling results suggest that collective epithelial migration can emerge in vitro from reciprocal biomechanical interactions between distinct cell states, rather than from leader-like cell behavior alone, and that such interactions can produce a predator-prey-like pursuit-escape mode of collective migration. Our model also provides a framework for investigating the biochemical and biomechanical regulation of collective cellular migration. It can be readily extended from in vitro to in vivo context and can incorporate the effects of substrate topology and soluble signaling factor-driven chemotaxis. Author summaryWhen epithelial cells repair a wound, they move as coordinated groups rather than as isolated individuals. Cells of different sizes may contribute in distinct but connected ways. We developed a computational model to examine how a large cell and neighboring regular-size cells move together. In the model, cells attach to the underlying surface and one another, form protrusions that set their direction, and redistribute the force-generating protein myosin after contact. We found that group motion depends on a balance among surface attachment, cell-cell adhesion, movement of regular-size cells toward the large cell, and myosin-driven turning of the large cell. Depending on this balance, a mixed-size cluster can travel along a nearly straight path or rotate persistently, exhibiting pursuit-and-escape-like interactions between the large cell and surrounding regular-size cells. Rotation occurs when the large cells turning effect outweighs the translational motion driven by regular-size cells, while cell-cell adhesion keeps the cluster together. Our results show that collective migration can emerge from reciprocal mechanical interactions between cells in different states, not only from a "leader" cell acting alone. The model also provides experimentally testable predictions for how cell size, adhesion, and internal myosin distribution shape cell cluster movement.

During cytokinesis in amphibian and echinoderm embryos, propagating waves of Rho activity and F-actin assembly - cortical excitability - are amplified and focused at the equatorial cortex by the mitotic spindle. Such cortical waves are proposed to result from fast positive feedback based on Rho and the Rho GEF, Ect2, and delayed negative feedback involving F-actin and the Rho GAP, RGA-3/4. Currently, this model is tenuous, in part because the relationship between F-actin and RGA-3/4 is not well understood. Therefore, we investigated how RGA-3/4 regulates F-actin structure both in vivo and in vitro. Full-length RGA-3/4, when expressed alone in Xenopus oocytes, localizes to and bundles cortical F-actin. In vitro, purified full-length RGA-3/4 protein directly binds to and bundles F-actin. TIRF microscopy revealed that RGA-3/4 binds to F-actin cooperatively and accumulates on the filaments before filament crosslinking events. Structure-function analyses demonstrated both the amino-terminal region and the intrinsically disordered region at the carboxyl-terminal portion of RGA-3/4 are essential for normal F-actin interaction. These results provide the first demonstration that RGA-3/4 is a direct, cooperative F-actin binding and bundling protein. We propose that its special features make RGA-3/4 uniquely suited to provide F-actin-dependent negative feedback during cortical excitability and cytokinesis.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

Aims: Mature, contractile vascular smooth muscle cells (vSMC) in muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Given the complicated embryonic origins and conflicting views on vSMC heterogeneity, our aim was to establish how closely hPSC-vSMC of a known germ layer lineage and defined phenotype are representative of vSMC obtained from aorta (AoSMC). Methods and Results: We generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines and performed phenotypic and mechanical comparisons with AoSMC. Synthetic hPSC-vSMC and cultured AoSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMC are non-proliferative, capable of phenotype switching and have elevated levels of contractile proteins (CNN1, TAGLN, ACTA2). These contractile proteins also displayed density-dependent, cell-to-cell heterogeneity in both AoSMC and hPSC-vSMC. Single-cell traction force measurements on micropost array detectors (mPADs) yielded comparable forces per micropost in the nN range across cell types. Quantitative 3D force measurements on humanized smooth muscle cell microtissues (SMTs) developed similar forces per cell. Larger forces were generated by MEKi-treated hPSC-vSMC and AoSMC versus controls. Conclusions: We have systematically studied the properties of hPSC-vSMC and performed comprehensive phenotypic and mechanical comparisons with AoSMC. These results demonstrate that synthetic hPSC-vSMC generated in vitro and cultured AoSMC derived from tissues are comparable and mechanically similar, but results with hPSC-vSMC are more reproducible.

The human genome encodes more than 60 proteins containing Rho GTPase activating protein (RhoGAP) domains, many of which remain understudied with respect to their target specificity and biological roles. SYDE1 and SYDE2 are two such orphan RhoGAPs, for which there are few studies characterizing their biochemical and cellular functions and conflicting reports identifying their cognate GTPases. We previously identified SYDE1 and SYDE2 in a screen for substrates of the c-Jun N-terminal kinases. Here, we show that SYDE1 and SYDE2 are preferentially phosphorylated by JNK1 relative to other mitogen-activated protein kinases (MAPKs) at sites proximal to a kinase docking region. Purified SYDE1 and SYDE2 are shown to have significant catalytic GAP activity toward RhoA, Rac1, and Cdc42. However, neither up- nor down-regulation of SYDE1/2 expression leads to detectable changes in bulk GTP loading of any of these GTPases. Nevertheless, we demonstrate that SYDE1 and SYDE2, in a partially GAP-dependent manner, increase cell spreading and number of focal adhesions, and promote more directionally persistent migration in HEK293 cells. Together, these findings establish SYDE1 and SYDE2 as robust JNK substrates with catalytic activity toward a set of Rho GTPases and reveal basic functions of SYDE1 and SYDE2 in regulating cell morphology, adhesion, and migration.

Focal adhesions maintain force-bearing attachment between cells and the extracellular matrix but can also undergo dynamic remodeling. Their assembly and actomyosin tension are coupled through mechanochemical feedback. The processes underlying this feedback are not instantaneous and therefore involve a time delay. However, how this delayed feedback gives rise to stable adhesion maintenance or dynamic remodeling remains unclear. Here, paired time-lapse measurements of vinculin fluorescence and traction stress revealed distinct local adhesion-force dynamics, including low-fluctuation and recurrent fluctuation patterns. To examine how these patterns could arise, we formulated a minimal mechanochemical model coupling focal adhesion assembly and actomyosin force through delayed reciprocal feedback. The model exhibited stable and oscillatory modes depending on feedback strength, the balance of opposing feedback effects, and the effective feedback delay. Bistability and hysteretic switching also occurred in a subset of parameter space, and the oscillation period followed a power-law relation with the delay. These results suggest that stable adhesion maintenance and dynamic remodeling can emerge from a common mechanochemical feedback architecture.

Actin cross-linkers are essential modulators of the actin cytoskeleton, enabling structural diversity and dynamic remodeling. Among them, calcium/calmodulin-dependent protein kinase II (CaMKII) is unique in serving a dual role as a kinase and as a multivalent structural binder of actin in dendritic spines, where it plays a major role in supporting dendritic spine structure via its {beta} subunit. To understand the basis of the structural function of CaMKII, we quantified the morphology and mechanics of actin bundles formed by CaMKII and directly compared them with bundles assembled by the canonical bivalent cross-linkers -actinin and fascin. Using fluorescence microscopy, we measured contour length, straightness ratio, and persistence length across different cross-linker concentrations. Fascin generated progressively shorter, straighter, and stiffer bundles with increasing concentration, whereas -actinin bundles remained largely unchanged. In contrast, increasing CaMKII concentration reduced both bundle straightness and persistence length, revealing a unique concentration-dependent increase in bundle flexibility. To investigate the structural origins of these mechanical behaviors, we complemented the experiments with coarse-grained simulations of cross-linked actin bundles. Simulations revealed that CaMKII-generated bundles retained a substantially larger fraction of their curvature in their time-averaged configuration than bundles formed by -actinin or fascin, indicating that bundle mechanics are strongly influenced by organizational features beyond thermal bending fluctuations alone. CaMKII bundles displayed behavior consistent with additional structural complexity arising from their multivalent architecture and flexible linker domains. Together, our experimental and computational results identify CaMKII as a structurally and mechanically distinct actin cross-linker. We propose that the combination of multivalency and linker flexibility enables CaMKII to function as a molecular structural pivot, promoting flexible, adaptable actin assemblies that are mechanically compatible with the dynamic remodeling required for dendritic spine plasticity.

During cell migration, branched actin filaments nucleated by the Arp2/3 complex induce leading edge protrusions, whereas directionality of cell migration is controlled by microtubules. We showed previously that Adenomatous Polyposis Coli (APC) initiates branched actin assembly at microtubule tips, which can explain how microtubules control directional protrusion. Here, we investigate a link from APC to Arp2/3 complex activity. We show that protrusion-generating activity of APC resides in its N-terminal region containing the Armadillo Repeat Domain (ARD). Furthermore, Asef1/ARHGEF4, a Cdc42 GEF known to be activated by the ARD of APC, as well as Cdc42 itself and its effector N-WASP, an Arp2/3 complex activator, are all required for the assembly of branched actin filaments in neuronal growth cones and neurite outgrowth. As downregulation of Asef1, Cdc42 or N-WASP produces phenotypes similar to APC knockdown, we propose that microtubules regulate directional cell migration and neuron navigation by inducing local membrane protrusion through the APC - Asef1 - Cdc42 - N-WASP - Arp2/3 pathway. These findings bridge a significant gap in our knowledge of cell migration.

ABSTRACT Objective: Skeletal muscle growth rate differs substantially among diverse pig breeds. In the present study, RNA-seq was performed to identify differentially expressed genes (DEGs) across pig breeds with distinct growth rates. CCND2 showed obvious differential expression among pig breeds. Although CCND2 is essential for cell cycle control, its biological function in skeletal muscle differentiation has rarely been reported. This work aims to identify DEGs in pig breeds with different growth rates and explore CCND2 role in porcine skeletal muscle development. Methods: RNA-seq was performed to identify differentially expressed genes (DEGs) across pig breeds with distinct growth rates. Weighted gene co-expression network analysis (WGCNA) was used to identify gene modules potentially associated with skeletal muscle growth and development. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed to validate the role of CCND2 in skeletal muscle differentiation. Results We identified 5,416, 4,846, and 2,777 DEGs in the Duroc pig (DU) vs. Jiangquan black pig (JQ), DU vs. Laiwu pigs (LW), and JQ vs. LW comparison groups, respectively. WGCNA identified two gene modules (lightgreen and darkmagenta) potentially associated with skeletal muscle growth and development. CCND2 exhibited differential expression in comparisons between DU vs JQ and DU vs LW, with higher expression observed in Duroc pigs. qRT-PCR analysis revealed that overexpression of CCND2 at day 6 (D6) of cell differentiation significantly increased the expression levels of CCND2 gene and markers of myogenic differentiation (p < 0.05). Conversely, knockdown of CCND2 significantly decreased the expression levels of CCND2 gene and myogenic differentiation markers (p < 0.05). Conclusions This study identified breed-specific DEGs and key gene modules in porcine skeletal muscle and verified that CCND2 promotes myogenic differentiation. These findings provide a genetic theoretical basis for further investigations into skeletal muscle growth and development. Keywords pig, skeletal muscle, transcriptome, WGCNA, CCND2

Wound repair is an important mechanism to preserve tissue integrity in organisms after injury. However, why different tissues exhibit different mechanisms to repair wounds is a long-standing question that remains unanswered. In this work, we theoretically explore the role of the purse string, an actomyosin contractile cable used by tissues to close small wounds. Does the tissue 3D geometry influence the efficiency of the purse string in driving wound closure? Using a 3D biophysical model, we study in silico tissues with the same cell volumes but different aspect ratios, ranging from squamous to thick and tall tissues. The model predicts that taller cells are easily deformed by the purse string. In contrast, very squamous cells require a very strong purse string that might demand additional cellular mechanisms to close the gap. These findings establish a theoretical framework to predict the optimal biophysical mechanisms of wound healing in different tissues. Graphical abstractCells of different aspect ratios can be observed in a range of organisms with different function and mechanics. The wound healing efficiency of the purse string increases with the cell aspect ratio in our theoretical exploration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/743165v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@d44ab0org.highwire.dtl.DTLVardef@1737cbaorg.highwire.dtl.DTLVardef@101b5d4org.highwire.dtl.DTLVardef@1487f26_HPS_FORMAT_FIGEXP M_FIG C_FIG

Activated leukocyte cell adhesion molecule A (Alcama) plays a role in axonal guidance, cell differentiation, and retinal lamination in a developing retina and was identified as a marker for activated Muller glial cells in adult zebrafish. However, its spatiotemporal localization and its involvement in retina regeneration remains unclear. Here we induced focal photoreceptor damage in zebrafish using laser photocoagulation and examined the expression and localization of Alcama at different time points post lesion. Immunohistochemistry in wild type fish and Tg(kdrl-EGFP) fish showed Alcama localized to the blood retina barrier with increased expression in Muller glial end feet and radial processes in a regenerating retina. To confirm its role in retina regeneration, alcama expression was transiently knocked down using morpholinos in adult fish. Scanning laser ophthalmoscopy, Zpr1 immunostaining and EdU staining showed delayed retina regeneration in alcama knockdown fish, indicating a possible role for Alcama in zebrafish retina regeneration.

Adult zebrafish efficiently regenerate skeletal muscle following different types of injury; however, the molecular programs involved in repair after extensive cryoinjury remain to be comprehensively characterized. Here, we explored the transcriptomic response of adult zebrafish skeletal muscle at 7 days post cryoinjury (dpci), a stage marked by ongoing tissue clearance, progenitor expansion, myogenic differentiation, and nascent myofiber formation, and compared it with phase-matched stab wound injury. Cryoinjury induced a broad transcriptional response, with 5,330 differentially expressed genes. Integrated enrichment and protein-protein interaction analyses revealed that, at 7 dpci, zebrafish skeletal muscle functions as an integrated regenerative system in which immune remodeling, progenitor expansion, myogenic differentiation, extracellular matrix reconstruction, mechanotransduction, biosynthetic adaptation, proteostasis, and intracellular trafficking operate simultaneously. In parallel, mature sarcomeric and oxidative metabolic programs were suppressed, consistent with ongoing tissue reconstruction and structural immaturity. Comparison with stab-wounded skeletal muscle revealed substantial transcriptional conservation, as 612 of 717 stab-wound-responsive genes (85%) were also differentially expressed after cryoinjury. Shared upregulated genes formed coherent functional modules related to proliferation, extracellular matrix organization and signaling, immune regulation, muscle differentiation, and protein processing. Thus, distinct injury modalities converge on a common regenerative program, while cryoinjury elicits a quantitatively broader transcriptional response. These findings support a conserved regenerative architecture of adult zebrafish skeletal muscle repair, in which interconnected biological modules act coordinately, with the breadth of transcriptional engagement reflecting regenerative demand.