BMC Molecular and Cell Biology

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Androglobin (ADGB), a protein essential for spermatogenesis, is the most structurally unusual member of the vertebrate globin superfamily. It combines a calpain-like domain with a circularly permuted globin domain containing an embedded calmodulin-binding IQ motif, an architecture suggesting complex regulatory functions that remain poorly understood. Here, we investigated whether ADGB undergoes calcium-dependent post-translational processing, like it has been described for other calpains. ADGB underwent robust proteolytic processing upon calcium stimulation, generating several stable cleavage products following ectopic expression in mammalian cells. In vitro proteolysis assays demonstrated that ADGB cleavage requires cytoplasmic factor(s) and is strongly enhanced by Ca2+. While this process is sensitive to pan-calpain inhibition, siRNA-mediated knockdown excluded calpain 1 (CAPN1) and calpain 2 (CAPN2) as primary mediators of ADGB cleavage. In contrast, depletion of the calpain small regulatory subunit CAPNS1 markedly reduced calcium-dependent ADGB proteolysis, implicating a CAPNS1-associated calcium-responsive proteolytic pathway. Domain-mapping analyses localized the major cleavage hotspot between the N-terminal calpain-like domain and the globin-containing C-terminal region, indicating that proteolysis separates the protease-like and globin modules of the ADGB chimera. The isolated globin domain displayed enhanced interaction with calmodulin compared with full-length ADGB, whereas the extended C-terminal region impeded this interaction. Furthermore, unlike full-length ADGB, the isolated globin domain exhibited preferential localization to centrosomal structures. Collectively, these findings identify calcium-dependent proteolysis and altered subcellular localization of the isolated globin domain as previously unrecognized properties of ADGB that may be relevant to its role in ciliary biology.

Cell migration depends on coordinating cell shape changes with force generation, yet how these processes are integrated remains unclear. Here, we combine live-cell imaging with traction force microscopy and computational analysis to quantify cell morphology, motility and force generation in migrating fibroblasts. We find that traction force magnitudes display a multimodal distribution, suggesting discrete migratory regimes. Using a Hidden Markov Model, we identify distinct force states that exhibit differences in shape and motion metrics, and show that individual cells transition between force states over time. To test the role of cytoskeletal organization in establishing the identified states, we analyzed cells lacking Arpc2, which disrupts branched actin assembly. Despite reduced forces and altered morphology, these cells also exhibit three migratory states. State transitions occur more frequently in cells lacking Arpc2 and unlike normal cells their protrusion geometry is force dependent. Together, our findings show that cell migration is organized into discrete mechanical states that couple morphology, motility and force generation. SUMMARY STATEMENTFibroblast motility involves distinct migratory states. These states exist independent of branched actin. However, state transition frequencies, traction force magnitudes and protrusion geometry are branched actin dependent.

Microtubule-destabilizing agents (MDAs) and microtubule-stabilizing agents (MSAs) are commonly used chemotherapeutic agents due to its activity to induce cell death by compromising the dynamics of spindles during mitosis. Ewing sarcoma, the second most common pediatric bone cancer, is known to selectively respond to MDAs as a first-line treatment, but not to MSAs. Ewing sarcoma cells carry an aberrant EWSR1-FLI1 fusion gene and only one wildtype EWSR1 allele. To investigate the origin of this MDA sensitivity, we used an (AID-EWSR1/wt: EWSR1-FLI1-mCherry/wt) cell line that enables conditional induction of EWSR1-FLI1 expression (Tet-On system) and EWSR1 knockdown derived from one EWSR1 allele (auxin-degron system). A combination of EWSR1-FLI1 expression and EWSR1 knockdown induces apoptosis upon nocodazole treatment, a type of MDA. Our study revealed that the mitotic spindles of Ewing sarcoma cells (A673, RD-ES and SK-ES1) contain elevated levels of tubulin damage, visualized with GTP-tubulin, compared to mesenchymal stem cells (MSC). Consistently, the combination of EWSR1-FLI1 expression and EWSR1 knockdown in (AID-EWSR1/wt: EWSR1-FLI1-mCherry/wt) cell line also leads to an increased incidence of damage in mitotic spindles. Together, we propose that the sensitivity of Ewing sarcoma cells is derived from the increased levels of damage to mitotic spindles caused by EWSR1-FLI1 expression and EWSR1 knockdown.

Ewing sarcoma is a pediatric cancer that develops in skeletal elements. The majority of Ewing sarcoma patients carry the aberrant EWSR1-FLI1 fusion gene. Despite trisomy 8 being an additional common aberration associated with a poor prognosis for patients, its induction mechanism remains unknown. When the EWSR1-FLI1 gene is formed, the cell loses one wildtype EWSR1 allele. To elucidate the induction mechanism of trisomy 8, we generated a cell line that allows for the conditional induction of EWSR1-FLI1 expression and EWSR1 knockdown (derived from a single EWSR1 allele. Specifically, the conditional cell line was generated by integrating the Tet-on EWSR1-FLI1 construct into the AAVS locus and adding a miniAID tag at the 5 end of the EWSR1 locus using auxin-degron system. A combination of the EWSR1-FLI1 expression and degradation of one allele-derived EWSR1 induced a high incidence of trisomy 8 within eight days, enhancing colony formation. Mechanistically, trisomy 8 is induced by the haploinsufficiency of EWSR1, and the remaining EWSR1 proteins are likely inhibited by interaction with EWSR1-FLI1. Our data showed that the knockout of EWSR1 alone was sufficient to increase the incidence of trisomy 8. Expression of wild-type EWSR1 in EWSR1 knockout cells rescued the high incidence of trisomy 8. In contrast, the EWSR1:R565A mutant, which lacks the ability to interact with Aurora B kinase, failed to rescue this phenotype. We propose that the combination of EWSR1-FLI1 expression and loss of EWSR1 contributes to the induction of trisomy 8 through the compromised EWSR1-Aurora B pathway. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/726567v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@999891org.highwire.dtl.DTLVardef@1ef6748org.highwire.dtl.DTLVardef@65e475org.highwire.dtl.DTLVardef@179da40_HPS_FORMAT_FIGEXP M_FIG C_FIG

Immortalized cell lines are widely used in biological research despite their known differences from their tissues and cell types of origin. Such cell lines are especially popular for testing hypotheses regarding the activity of cis-regulatory elements (CREs) that regulate gene expression. Previous investigations of blood and skin cell lines revealed many differences between the transcriptional regulatory networks of the cell lines and the associated primary cells. Similar comparisons for other tissues have been limited. Here, we used ATAC-seq to profile CREs in four immortalized liver cell lines and found many differences between each cell lines CREs and primary liver tissue, including differences in the transcription factors that are likely to bind them and differences in the genes that they are likely to regulate. Modifying cell culture conditions based on recommendations in the literature did not improve the similarity with primary liver tissue. Our results suggest that differences between the transcriptional regulatory networks in cell lines and primary tissue should be considered when designing and interpreting cell line experiments.

Many transcription factors (TFs) bind only a subset of their canonical binding sites in mammalian cells. To identify differences between bound and unbound sites we examined Zta(N182S), a mutant of the Epstein Barr Virus (EBV)-encoded Zta bZIP protein that binds distinct DNA sequences that are not strongly bound by any known human or viral TF, reducing the effects of selective pressure on endogenous genomic binding sites. We stably expressed Zta(N182S) in human HEK293 cells and monitored protein binding (ChIP-seq) and effects on chromatin accessibility (ATAC-seq). Zta(N182S) binds ~10% of the 14,979 genomic occurrences of the canonical 9-mer ATCACTCAT, creating stronger overall ATAC-seq signal compared to control cells, suggesting nucleosome displacement. Nucleosome occupancy, either predicted or experimentally determined (MNase), indicates that canonical Zta and Zta(N182S) sites are more strongly bound when they are ~60bp from a positioned nucleosome dyad. These data suggest that Zta and Zta(N182S) binding results in nucleosome remodeling, consistent with pioneer-like activity. Examination of amino acids across Zta and human bZIPs identifies four conserved basic amino acids, a proline, and acidic amino acids immediately N-terminal of the basic amino acids of the bZIP domain (PARRTRKPQQPESLEECDSELEIKRYKN). We term this new protein motif "BPabZIP" (Basic-Proline-acidic bZIP). Molecular structure predictions for both Zta and human Fos/Jun reveal the basic amino acids interacting with the acidic patch on the nucleosome. The acidic amino acids act as an a-helical extension of the basic region that mimics DNA by interacting with histones H2A and H2B. Taken together, our analyses of this synthetic TF reveal a pioneer-like mechanism that is present in both human and viral bZIP proteins.

Proper maintenance of gene expression in response to mutations or environmental fluctuations is critical for cell development and survival. Recently, a novel genetic compensation mechanism was described wherein mutant mRNA decay triggers increased transcription of paralogous genes. This effect was reported for several genes, including {beta}-actin (Actb) in mouse embryonic stem cells, where Actb mRNA with a premature termination codon enhances transcription of its paralog, {gamma}-actin (Actg1), and partially rescues cytoskeletal defects. Here we show that, in both mouse and human embryonic stem cells, mutations in the ACTB gene, regardless of mutant mRNA expression, trigger genetic compensation. Furthermore, transgenic expression of mutant ACTB mRNA with a premature stop codon fails to induce genetic compensation. Depletion of the SRF or MRTF-A transcription factors, which are known to increase ACTB transcription in response to low ACTB protein levels, diminishes the genetic compensation response in ACTB mutants. These results suggest that genetic compensation in ACTB mutants is primarily mediated by a transcriptional feedback loop via SRF/MRTF-A, independent of the expression or degradation of mutant ACTB mRNA.

Protein homeostasis - i.e., proteostasis - is the biological process by which the qualitative and quantitative balance of the proteome is conducted, either by preserving functionally relevant proteins or by degrading unnecessary ones. Stress conditions can modulate cellular proteostasis in order to promote cytoprotection and preserve the viability of living organisms. Among the cellular pathways already described that can play an important role in preserving biological functions by modulating proteostasis are the heat shock response and the ribosome quality control pathways. In this work, we show that the Rqc1p protein is necessary for the thermoadaptation of S. cerevisiae to heat shock, as RQC1-deficient yeast is sensitive to elevated temperatures. In silico approaches - such as multiple sequence alignment, structural analysis, and molecular dynamics simulations - confirmed earlier predictions that Rqc1p shares characteristics with the bHLH family of proteins. We also verified, through computational prediction of sub-cellular localization, that S. cerevisiae Rqc1p contains nuclear localization signals, suggesting that this protein can potentially be translocated toward the nucleus, thereby broadening its current range of recognized biological functions in this organism. Also, analysis of yeast transcriptomes subjected to heat shock showed that Rqc1p mRNA levels do not fluctuate in response to heat shock, suggesting that cellular concentrations of Rqc1p are already at optimal levels to elicit a rapid and effective response during thermal stress in S. cerevisiae.

Hypoxia and anoxia are known to suppress cell proliferation due to an increase in replication stress and activation of DNA damage checkpoints. Embryos of the annual killifish Austrofundulus limnaeus show a strong tolerance to extended anoxic exposure, indicating an improved genomic stability under oxygen starvation. Here we investigate the cell cycle regulation of the anoxia tolerant killifish embryonic cell line PSU-AL-WS40NE during anoxic exposure. Live cell imaging confirms continued cell proliferation of WS40NE cells for the first 24 hours of anoxic exposure with minimal cell death. Fluorescent imaging shows that cells begin to accumulate in G1 after the first day in anoxia with a pronounced and rapid entry into the S phase upon reoxygenation. Pharmacological inhibition tests show that this response appears to be reliant more on ATR signaling then ATM, suggesting that increased {gamma}H2AX levels are driven by increased replication stress instead of DNA damage. This conclusion is further supported by an apparent lack of induction of a G2 checkpoint in these cells suggesting that DNA damage during anoxic replication is minimal. Maintaining cellular proliferation during initial exposure to anoxia and accumulating cells in the G1 phase for extended anoxic exposure is likely one way that embryos of the annual killifish are able to survive prolonged anoxia and provides insight into mechanisms that enable cells to proliferate under metabolic stress.

Cells can enter a reversible non-proliferative state called quiescence either spontaneously or in response to nutrient deprivations. Metal ions are essential nutrients and play wide-ranging regulatory and signaling roles in biological processes. We previously showed that zinc is an essential nutrient for the mammalian cell cycle as zinc deprivation drives cells into quiescence, and this quiescent state is associated with changes in iron, copper, and manganese, suggesting broad remodeling of metal homeostasis. Here we examine whether metal remodeling is a general feature of quiescence by inducing quiescence via different triggers (zinc deficiency, serum starvation, and growth factor withdrawal) in MCF10A cells. Fluorescence microscopy and elemental analysis reveal significant trigger-dependent changes in the labile and total metal pools of quiescent cells. To gain insight into these differences, we carried out RNA sequencing and differential expression analysis, focusing on metal associated, metal regulatory, and metal homeostasis genes. While core quiescence pathways are shared across triggers, quiescence states remain molecularly distinct. A significant percent of 2458 metal homeostasis annotated genes are differentially expressed including 55% in starvation-induced quiescence, 50% in zinc deficiency-induced quiescence and 21% in growth factor withdrawal-induced quiescence. Our results also showed unique alteration of genes involved in major metal dependent processes including antioxidant activity, oxidative phosphorylation, heme metabolism, and chromatin accessibility in different quiescence states. Overall, this work demonstrates that metal homeostasis is systematically rewired during cellular quiescence with associated effects on genes that regulate critical biological processes. Significance StatementCells constantly evaluate their nutrient and energy status and integrate these signals into proliferation-quiescence decisions. Quiescence prevents cells from passing damage to daughter cells. Metals are essential micronutrients for biological processes. While limitation of zinc can drive cells into quiescence and alter other metals, how metals are remodeled and whether this is a common feature of quiescence was unknown. Here we examined three quiescence triggers by modifying growth media and serum, the primary source of metals. We report significant changes in labile and total Cu, Fe, Zn and Mn pools, and metal associated genes in response to distinct quiescence triggers. These changes converge on mitochondrial function, cellular antioxidant activity, and heme biosynthesis in a trigger specific manner.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

Keloids are fibroproliferative skin disorders arising following dermal injury that extend beyond the original wound margins. Their pathogenesis remains poorly understood, and current treatments are associated with high recurrence rates. Identifying transcriptomic biomarkers that distinguish keloids from other skin and scar phenotypes may provide insight into disease mechanisms and facilitate the development of targeted therapeutic approaches. However, previous transcriptomic studies have often been limited by small sample sizes, pairwise comparisons between tissue classes, heterogeneous data-integration strategies, and a reliance on conventional differential gene expression (DGE) analysis. Here, we employed a multi-stage machine learning (ML) workflow for robust keloid biomarker discovery using transcriptomic datasets derived from both bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq). We assembled and harmonized, to the best of our knowledge, the largest curated cross-study keloid transcriptomic cohort currently available, comprising 81 samples from 13 independent studies spanning four clinically relevant tissue classes: normal skin, normotrophic scar, hypertrophic scar, and keloid scar. Through study-aware cross-validation, feature selection, partition-stability analysis, and bootstrap validation across multiple ML classifiers, we identified a panel of eight highly consistent biomarkers capable of distinguishing keloid from non-keloid samples. These biomarkers were associated with dysregulation of extracellular matrix homeostasis, fibrosis-resolution pathways, vascular remodelling, and metabolic reprogramming. Comparison with conventional DGE analysis demonstrated substantial agreement while also highlighting important differences between the two approaches. In particular, FASN was consistently identified by the ML workflow as an upregulated discriminatory biomarker despite exhibiting weak, non-significant differential expression in the DGE analysis. Cell-type-specific analysis further supported this finding, revealing significant FASN upregulation in fibroblast and vascular endothelial populations. These results demonstrate that ML and DGE capture complementary aspects of transcriptomic variation. This study provides a robust strategy for cross-study transcriptomic biomarker discovery and identifies candidate genes and pathways for future mechanistic and therapeutic investigation in keloids. 1 Author SummaryKeloids are abnormal scars that continue to grow beyond the original wound and can be difficult to treat because they frequently recur after therapy. Although many studies have investigated the biology of keloids, the molecular mechanisms that distinguish them from other scar types remain incompletely understood. Identifying biomarkers involved in keloid formation may help inform improved treatment strategies. Previous transcriptomic studies have often been limited by small sample sizes and inconsistent analytical approaches. In this study, we combined gene-expression data from multiple independent studies to create, to the best of our knowledge, the largest cross-study transcriptomic collection available for keloid analysis. We then applied several machine learning approaches to identify genes that consistently distinguished keloids from other skin and scar phenotypes. The identified biomarkers were associated with extracellular matrix remodeling, fibrosis, vascular function, and cellular metabolism. One gene involved in fatty-acid synthesis, FASN, was repeatedly identified by the machine learning analyses despite being overlooked by conventional gene-expression methods. Additional single-cell analyses confirmed elevated FASN expression in specific cell populations within keloid tissue. More broadly, this work provides a strategy for discovering robust biomarkers from heterogeneous biological datasets and identifies molecular targets for future studies of keloid disease.

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

Microtubules are cytoskeletal structures composed of polymers of /{beta}-tubulin heterodimers. They play a central role in cell division and motility by a stochastic process of alternating polymerization and depolymerization episodes (dynamic instability) that can be modulated by phosphorylation. Protein kinase C and cyclin-dependent kinase 1 are known to phosphorylate Ser165 of -tubulin (:Ser165) and Ser172 of {beta}-tubulin, ({beta}:Ser172), respectively. Using all-atom molecular dynamics simulations of 6-mer {beta}-tubulin systems modeled on the cryo-EM structure of a microtubule (PDB 3J6E), the impact of phosphorylation at each site is explored in terms of secondary structures (:helix H8/loop T7 segment and {beta}:loops T3/T5) that lie at the inter-dimer cleft near the E-site {beta}:GTP. If properly aligned, :Glu254 (helix H8) hydrolyzes {beta}:GTP to GDP thereby triggering the transition from a polymerizing to a depolymerizing microtubule. -Tubulin phosphorylated at :Ser165 displaces helix H8 (:Glu254/:Gln256) and loop T5 towards the {gamma}-phosphate of {beta}:GTP. This movement coincides with a shift of the {beta}:GTP nucleotide by 4.5-5.5 [A], stabilization of the {gamma}P of {beta}:GTP by additional H-bonding and weakened inter-dimer interactions. In a phosphorylated {beta}:Ser172 system, loop T5 is displaced toward {beta}:GTP and coincides with stabilization of inter-dimer interactions. Therefore, phosphorylation of either - or {beta}-tubulin generates a distinct profile of intramolecular rearrangements that remodel the inter-dimer cleft and modulate dynamic instability. These profiles may provide a useful reference for screening mutations identified in tumor genomes.

Understanding cis-regulatory elements (CREs) at the single cell level is fundamental to deciphering transcriptional changes during development, cell differentiation, and homeostasis. Recent studies have shown that arbitrary peak-calling thresholds complicate data interpretation and cross-study comparisons. Furthermore, due to the inherent sparsity of single-nuclei ATAC-seq (snATAC-seq) data, distinguishing between truly inaccessible regions and technical dropouts remains challenging. Our analysis of snATAC-seq experiments performed in a well-established cell line suggests that the dichotomy between accessible (open) or inaccessible (close) CREs is misleading. Thousands of accessible regions are present in a very small fraction of cells of the population but they are repeatedly identified, suggesting that they have a low accessibility or are only transiently accessible. However, depending on the detection threshold selected they could be considered as either genuine CREs or noise. To resolve this inconsistency, we propose a model where chromatin accessibility is treated as a continuum, defined by a probability of accessibility (Pa) for each accessible region across cell types and conditions. Through computational simulations, we demonstrate that snATAC-seq results can be explained by a simple "balls into bins" probability model, offering a theoretical framework for calculating Pa distributions from any snATAC-seq dataset. Furthermore, we examine how Pa distributions shift following activation of the TGF{beta} signaling pathway. This probabilistic approach removes the reliance on arbitrary thresholds and supports a more quantitative, and dynamic understanding of accessible regions function.

Myosin 3A (MYO3A) is an unconventional myosin involved in the formation and maintenance of hair-cell stereocilia of the sensory epithelia in the inner ear. The kinase domain has been implicated in phosphoregulation of MYO3A activity through intermolecular autophosphorylation. Previous studies using mass spectrometry identified two potential phosphorylation sites in the motor domain. To investigate the regulatory roles of these sites, we generated glutamic acid point mutations in our mchr-MYO3A{Delta}K construct to mimic phosphorylation and assayed the constructs for their ability to tip-localize and influence filopodial density via transfection into COS7 cells. The phosphomimic constructs were less able to generate filopodia when compared to wildtype constructs. To gain a better understanding of the phosphoregulation of MYO3A, we transfected COS7 cells with mchr-MYO3A{Delta}K in combination with GFP-tagged full-length MYO3A (GFP-MYO3AFL), or GFP attached to just the kinase domain of MYO3A (GFP-MYO3AKIN). Coexpression of mchr-MYO3A{Delta}K with either construct resulted in decreased mchr-MYO3A levels at the tips of filopodia and fewer filopodia at the edge of the cell, compared to cells expressing mchr-MYO3A{Delta}K alone. This implies that the kinase domain does not require motor activity to contribute to phosphoregulation of MYO3A, and that MYO3A phosphoregulation may be influencing filopodia initiation. Informatic analyses and structural predictions suggest that the two phosphorylation sites in the motor domain inhibit actin/MYO3A interactions. Taken together, these analyses link MYO3A phosphorylation with the regulation of its ability to create actin protrusions such as filopodia and stereocilia.

Cilia and flagella contribute to cell morphogenesis in multiple organisms. In the parasite Trypanosoma brucei, the flagellum is attached along the length of the cell body and acts as a guide for cell division, while its motility function is required for the completion of cytokinesis. To tease apart the contributions of flagellum length and motility to trypanosome morphogenesis, we investigated the coiled-coil containing domain 40 (CCDC40 or FAP172) protein. Iterative Ultrastructure Expansion Microscopy (iU-ExM) revealed that CCDC40 is associated to the 96-nm repeats of the trypanosome axoneme. CCDC40 depletion by RNAi leads to loss of components from the dynein regulatory complex, inner dynein arms and radial spokes, resulting in disconnected microtubule doublets and disorganised axoneme structure, abrogating motility and resulting in flagella and cell bodies 2-3 times shorter than normal. We show for the first time that this short flagellum phenotype is associated to slower tubulin incorporation and premature acquisition of the maturation marker FLAM8 but not of the locking protein CEP164C. Surprisingly, these short and immotile trypanosomes grow and divide normally. We discuss the significance of these observations for trypanosome morphogenesis and division.

Cofilin is a key regulator of actin dynamics that, along with a myriad of other actin-binding proteins, controls the balance of F- and G-actin in numerous cell types. While prior structural studies of the cofilin-actin binding interface have delineated many critical interactions between cofilin and actin, the roles of some residues within the cofilin-actin binding interface remain poorly defined. In this study, we investigate the role of cofilin S119 in the cofilin-actin interaction. Despite its unique position within the cofilin-actin interface and its putative role as a phosphorylation site, relatively little direct evidence exists to define whether it plays an important role in cofilin-actin dynamics. Using site-directed mutagenesis, we demonstrate that mutation of S119 to aromatic amino acids (W, F, Y) results in cofilins with strong actin bundling activity in living cells. This activity can be countered by the incorporation of mutants that disfavor actin rod forming activity (R21Q). Mutation of S119 to phospho-mimic (E) and non-phosphorylated (A) residues either strongly inhibits (E) or modestly increases (A) actin bundling activity. Expression of the S119W mutant in neurons reveals its impacts on spine length and size, while FRAP studies show that its mobile fraction is intermediate between that of LifeAct and WT cofilin. Finally, it is shown that the strong actin bundling phenotype associated with S119W inhibits the progression of optogenetically induced apoptosis.

Mechanical cues from the extracellular matrix regulate cancer cell behavior, but how these inputs are translated into distinct nuclear signaling responses remains incompletely understood. This study examined whether Piezo1, a mechanosensitive ion channel, contributes to cytoskeletal remodeling and differential regulation of YAP and {beta}-catenin localization in breast cancer cells exposed to defined mechanical cues. MDA-MB-231 breast cancer cells were cultured on substrates of defined stiffness and analyzed after Piezo1 knockdown or pharmacological modulation of Piezo1, Src signaling, myosin II activity, and actin polymerization. Nuclear localization of YAP and {beta}-catenin was assessed by immunofluorescence imaging, cytoskeletal organization was evaluated using filamentous and globular actin staining, protein phosphorylation was analyzed by capillary electrophoresis-based immunoblotting, and cell migration was assessed using a wound-healing assay. Piezo1 knockdown reduced YAP nuclear localization and increased {beta}-catenin nuclear localization, while Piezo1 activation partially reversed these localization changes. Piezo1 knockdown also disrupted filamentous actin organization, and pharmacological disruption of actin polymerization produced similar effects on YAP and {beta}-catenin localization. Piezo1 knockdown selectively reduced YAP tyrosine phosphorylation without altering canonical Hippo-associated YAP serine phosphorylation, and inhibition of Src signaling produced effects similar to Piezo1 knockdown. Functionally, Piezo1 knockdown impaired stiffness-dependent cell migration. These findings support a role for Piezo1 in linking extracellular mechanical cues to cytoskeletal organization and differential regulation of YAP and {beta}-catenin localization in breast cancer cells. This work provides a framework for understanding how mechanosensitive ion-channel signaling may contribute to context-dependent nuclear signaling responses during cancer cell mechanotransduction.