Cytoskeleton (Hoboken, N.J.)最新文献

Auditory hair cells, which convert sound-induced vibrations in the inner ear into neural signals, depend on multiple actin populations for normal function. Stereocilia are mechanosensory protrusions formed around a core of linear, crosslinked F-actin. They are anchored in the cuticular plate, which predominantly consists of randomly oriented actin filaments. A third actin population is found near hair cell junctions, consisting of both parallel and branched filaments. Actin depolymerizing factor (ADF) and cofilin-1 (CFL1) proteins disassemble actin filaments and are required to regulate F-actin in stereocilia, but their effect on cuticular plate and junctional actin populations is unclear. Here, we show that loss of ADF and CFL1 disrupts the patterning of stereocilia into orderly bundles and that this phenotype correlates with defective development of the cuticular plate and junctional actin populations. ADF/CFL1 continue to regulate these actin populations in mature cells, which is necessary for long-term maintenance of hair cell morphology.

Fibroblasts, crucial for maintaining tissue homeostasis, significantly shape the tumor microenvironment (TME). In pancreatic cancer, a highly aggressive malignancy, cancer-associated fibroblast (CAF)/extracellular matrix (ECM) units dominate the TME, influencing tumor initiation, progression, and treatment responses. Palladin, an actin-associated protein, is vital for fibroblast structural integrity and activation, playing a key role in CAF/ECM functionality. Palladin interacts with cytoskeletal proteins such as alpha-actinin (α-Act) and can therefore regulate other proteins like syndecans, modulating cytoskeletal features, cell adhesion, integrin recycling, and signaling. In this review, we propose that targeting the palladin/α-Act/syndecan interaction network could modulate CAF/ECM units, potentially shifting the TME from a tumor-promoting to a tumor-suppressive state. In silico data and reported studies to suggest that stabilizing palladin-α-Act interactions, via excess palladin, influences syndecan functions; potentially modulating integrin endocytosis via syndecan engagement with protein kinase C alpha as opposed to syndecan binding to α-Act. This mechanism can then affect the distribution of active α5β1-integrin between the plasma membrane and known intracellular vesicular compartments, thereby influencing the tumor-suppressive versus tumor-promoting functions of CAF/ECM units. Understanding these interactions offers likely future therapeutic avenues for stroma normalization in pancreatic and other cancers, aiming to inhibit tumor progression and improve future treatment outcomes.

Mechanotransduction leads to a variety of biological responses including gene expression, changes in cell shape, migration, tissue development, and immune responses. Dysregulation of mechanotransduction is implicated in the progression of various diseases such as cardiovascular diseases and cancer. The actin cytoskeleton plays a crucial role in transmitting mechanical stimuli. Actin filaments, essential for cell motility and shape changes, respond to mechanical cues by remodeling, influencing gene expression via the linker of nucleoskeleton and cytoskeleton complex and mechanosensitive transcription factors. This study employs the dithiobis(succinimidyl propionate) (DSP)-micrococcal nuclease (MNase) proteogenomics method to explore the relationship between cellular mechanosensing, chromatin architecture, and the identification of proteins involved in mechanosensitive nucleocytoplasmic shuttling, revealing how actin polymerization affects chromatin and gene expression. We found that depolymerization of actin filaments by latrunculin B (Lat B) for 30 min is sufficient to alter open chromatin and identified core-binding factor subunit beta as mechanosensitive nucleocytoplasmic shuttling protein.

FtsZ forms a ring-like assembly at the site of division in bacteria. It is the first protein involved in the formation of the divisome complex to split the cell into two halves, indicating its importance in bacterial cell division. FtsZ is an attractive target for developing new anti-microbial drugs to overcome the challenges of antibiotic resistance. The most potent inhibitor against FtsZ is PC190723, which is effective against all strains and species of Staphylococcus, including the methicillin- and multi-drug-resistant Staphylococcus aureus and strains of Bacillus. However, FtsZs from bacteria such as E. coli, Streptococcus, and Enterococcus were shown to be resistant to this inhibitor. In this study, we provide further evidence that the three pairwise bridging interactions, between residues S227 and G191, R307 and E198 and D299 and R202, between S7, S9, S10 β-strands and the H7 helix occlude the inhibitor from binding to E. coli FtsZ. We generated single, double and triple mutations to disrupt those bridges and tested the effectiveness of PC190723 directly on Z-ring assembly in vivo. Our results show that the disruption of S227-G191 and R307-E198 bridges render EcFtsZ highly sensitive to PC190723 for Z-ring assembly. Ectopic expression of the double mutants, FtsZ S227I R307V results in hypersensitivity of the susceptible E. coli imp4213 strain to PC190723. Our studies could further predict the effectiveness of PC190723 or its derivatives towards FtsZs of other bacterial genera.

Microtubules (MTs) are dynamic cytoskeletal filaments with highly conserved sequences across evolution, polymerizing by the GTP-dependent assembly of tubulin subunits. Despite the sequence conservation, MT polymerization kinetics diverge quantitatively between vertebrate brain, the model plant Arabidopsis and the protozoan Plasmodium. Previously, tubulin purified from seedlings of the plant Vigna sp. (mung) by temperature cycling was found to have a very low critical concentration. However, the lengths of MTs were sub-micron, much shorter than brain tubulin filaments. This was explained in simulations to be the result of the collective effect of high nucleation and GTP hydrolysis rates. Here, we test the effect of GTPase rates of affinity-purified Vigna sp. tubulin on microtubule polymerization and elongation. Affinity-purified mung tubulin is active and has a critical concentration of .37 μM. The GTP-dependent polymerization kinetics are transient, consistent with previous results. Polymerization is stabilized in the presence of either GTP analog GMPPNP (non-hydrolyzable) or GMPCPP (slow-hydrolyzable). Using interference reflection microscopy (IRM) we find polymerization with the non-hydrolysable analog significantly increases filament numbers, while lengths are unaffected for both GTP analogs. However, prolonged incubation with slow-hydrolyzable GMPCPP results in long filaments, pointing to GTP hydrolysis as a key factor determining MT length. We find the average GTPase turnover number of mung tubulin is 22.8 min^-1, compared to 2.04 min^-1 for goat brain tubulin. Thus modulating GTPase rates affects both nucleation and elongation. This quantitative divergence in kinetics despite high sequence conservation in the GTPase domains of α- and β-tubulin could help better understand the roles of selective pressure and function in the diverse organisms.

Epithelial-to-mesenchymal transition (EMT) is a key process where cells lose their adhesion properties and augment their invasive properties. α-Actinin4 (ACTN4) is an actin crosslinking protein that responds to mechanical stimuli and is found to be elevated in breast cancer patients. While ACTN4 has been implicated in regulating cancer invasiveness by modulating cytoskeletal organization, its nuclear functions remain much less explored. Here we address this question by first establishing a correlation between nuclear localization and invasiveness in breast cancer cells. Using cancer databases, we then establish a correlation between ACTN4 expression and EMT in breast cancer. Interestingly, TGFβ-induced EMT induction in MCF10A normal mammary epithelial cells leads to increased ACTN4 expression and nuclear enrichment. We then show that ACTN4 knockdown in MDA-MB-231 breast cancer cells, which harbor sizeable fraction of nuclear ACTN4, leads to reduced invasiveness and loss of mesenchymal traits. Similar behavior was observed in knockdown cells expressing K255E ACTN4, which is primarily localized to the cytosol. Together, our findings establish a role for nuclear ACTN4 in regulating invasiveness via modulation of EMT.

Most of the single point mutations of the LMNA gene are associated with distinct muscular dystrophies, marked by heterogenous phenotypes but primarily the loss and symmetric weakness of skeletal muscle tissue. The molecular mechanism and phenotype-genotype relationships in these muscular dystrophies are poorly understood. An effort has been here to delineating the adaptation of mechanical inputs into biological response by mutant cells of lamin A associated muscular dystrophy. In this study, we implement engineered smooth and pattern surfaces of particular young modulus to mimic muscle physiological range. Using fluorescence and atomic force microscopy, we present distinct architecture of the actin filament along with abnormally distorted cell and nuclear shape in mutants, which showed a tendency to deviate from wild type cells. Topographic features of pattern surface antagonize the binding of the cell with it. Correspondingly, from the analysis of genome wide expression data in wild type and mutant cells, we report differential expression of the gene products of the structural components of cell adhesion as well as LINC (linkers of nucleoskeleton and cytoskeleton) protein complexes. This study also reveals mis expressed downstream signaling processes in mutant cells, which could potentially lead to onset of the disease upon the application of engineered materials to substitute the role of conventional cues in instilling cellular behaviors in muscular dystrophies. Collectively, these data support the notion that lamin A is essential for proper cellular mechanotransduction from extracellular environment to the genome and impairment of the muscle cell differentiation in the pathogenic mechanism for lamin A associated muscular dystrophy.

Profilin is an actin monomer-binding protein whose role in actin polymerization has been studied for nearly 50 years. While its principal biochemical features are now well understood, many questions remain about how profilin controls diverse processes within the cell. Dysregulation of profilin has been implicated in a broad range of human diseases, including neurodegeneration, inflammatory disorders, cardiac disease, and cancer. For example, mutations in the profilin 1 gene (PFN1) can cause amyotrophic lateral sclerosis (ALS), although the precise mechanisms that drive neurodegeneration remain unclear. While initial work suggested proteostasis and actin cytoskeleton defects as the main pathological pathways, multiple novel functions for PFN1 have since been discovered that may also contribute to ALS, including the regulation of nucleocytoplasmic transport, stress granules, mitochondria, and microtubules. Here, we will review these newly discovered roles for PFN1, speculate on their contribution to ALS, and discuss how defects in actin can contribute to these processes. By understanding profilin 1's involvement in ALS pathogenesis, we hope to gain insight into this functionally complex protein with significant influence over cellular physiology.