The simple nematode Caenorhabditis elegans has long served as a powerful genetic model system for studying muscle structure and function, with sarcomeres of the striated body wall muscle (BWM) that are considered largely homologous to those in vertebrates. However, we have observed a population of F-actin in BWM separate from I-bands and centered at the sarcomere M-line, something not seen in vertebrate sarcomeres. Here, we investigate whether M-line-associated F-actin is a true feature of worm muscle, or an artifact of sample preparation. Using thin filament components tagged with fluorescent proteins, we confirm that living C. elegans BWM has a traditional sarcomere organization with actin restricted to I-bands, whereas two methods of fixation resulted in varying degrees of mislocalization of actin as well as barbed end capping protein/CapZ and tropomodulin. Additionally, we observe in live animals that α-actinin-rich dense bodies are flanked by discrete patches of CapZ, something we cannot observe in fixed animals, but which provides some support for a model in which dense bodies anchor thin filaments in a manner similar to vertebrate Z-discs. Overall, our work highlights the susceptibility of thin filaments in worm BWM to fixation artifacts and the potential utility in studying sarcomere structure in live animals.
Mammals have four tropomyosin (TPM) genes (TPM1, TPM2, TPM3, and TPM4) that produce various isoforms through alternative splicing. TPM1 generates both myofibrillar (Tpm1.1, Tpm1.2) and non-myofibrillar isoforms, including those with an exon 9d peptide or with an exon 9a peptide. These non-myofibrillar isoforms are involved in regulating actin-based structures and processes in non-muscle cells. This study focused on the expression of the non-myofibrillar HMW Tpm1 isoforms (Tpm1.3, Tpm1.4, Tpm1.5, Tpm1.6, Tpm1.7, and Tpm1.14) during human inducible pluripotent stem cells (hiPSC) differentiation into cardiomyocytes (CMs) at different time points (Days 0, 5, 10, 15, 20). We have determined the expression of various Tpm1 transcripts by qRT-PCR using isoform-specific primer-pairs. Western blotting with Tpm1-exon 6a and Tpm1-exon 9d antibodies and 2D Western blotting with Tpm1-exon 6a antibody followed by mass spectra analyses were used to evaluate protein expression. Transcripts of non-myofibrillar Tpm1 isoforms peaked at Day 15 and continued at a slightly lower level in mature hiPSC-CM until Day 20. However, no expression of Tpm1.3 or Tpm1.14 has been observed. Protein expression of Tpm1.4, Tpm1.5, Tpm1.6, and Tpm1.7 increases up to Day 15 but practically disappears by Day 20 hiPSC-CM, suggesting their production is decreased, or more likely they are degraded intracellularly. The results suggest that the proteins have the potential to be transiently involved in the early stages of CM differentiation. This study also demonstrates that Tpm1.5, when fused with YFP in an expression construct and transfected into embryonic chicken CM and Day 20 mature hiPSC-CMs, can be organized into cardiac myofibrils despite being previously characterized as a non-muscle isoform. Our observation is further substantiated by the fact that YFP-Tpm1.5 fusion protein can be ectopically expressed and incorporated into the myofibrils of chicken myotube skeletal muscle, which is known to be more stringent than cardiac muscle with regards to myofibril remodeling. Paradoxically, anti-6a antibody fails to recognize the organized YFP-Tpm1.5 fusion protein in embryonic chicken CMs, embryonic chicken skeletal muscle myotubes, or in Day 20 mature hiPSC-CMs. Interestingly, the antibody recognizes the denatured YFP-Tpm1 fusion protein in Western blot analyses. It is well-documented in the literature that an antibody epitope may fail to recognize its antigen when the antigen is in its native state in living cells, often because the epitope is buried, altered conformationally, or inaccessible. Conversely, while the Tpm1.Ex6a antibody stains Tpm1.5 in cell nuclei, the YFP-Tpm1.5 fusion protein does not localize to the cell nuclei. This suggests conformational differences between nuclear and cytoplasmic Tpm1 protein(s) and differential access of the fusion protein to different cellular locations. This also suggests that it is endogenous Tpm1 6a-containing protein identified in the nuclei.
Collective cell migration relies on coordinated cytoskeletal remodeling, yet the impact of live-cell actin filament probes on these dynamics remains poorly characterized. Here, we systematically compared the performance and cellular effects of fluorogenic jasplakinolide-based probes, SiR-actin and SiR-XActin, with the genetically encoded Lifeact reporter in epithelial monolayers. Using an injury-free wound healing assay combined with widefield images, particle image velocimetry, kymographs, and FUCCI-based cell cycle tracking system, we assessed how probe choice, efflux inhibition, and genetic modification influence actin organization, migration, and proliferation. SiR-XActin provided robust labeling of actin filaments with minimal perturbation in migration rate, directionality, or cell cycle progression, enabling stable visualization of cytoskeletal dynamics in monolayers. In contrast, SiR-actin and Lifeact produced either probe- or cell line-specific effects on migration persistence and wound closure, particularly under prolonged imaging. The use of verapamil, a commonly used phenylalkylamine-derived calcium channel blocker, improved probe retention and migratory persistence without altering proliferation. In contrast, FUCCI-expressing monolayers showed impaired motility independent of probe type, potentially reflecting cytoskeletal constraints associated with cell-cycle reporters. Lifeact-expressing cells exhibited an initial increase in migration rate followed by an incomplete wound closure, consistent with mild actin stabilization. Together, these findings identify SiR-XActin as a minimally perturbative, high-fidelity probe for monitoring actin filament dynamics during collective epithelial migration and highlight the importance of evaluating probe-cell compatibility for live cytoskeletal studies.
The technique of fluorescence resonance energy transfer (FRET) has been used in this study to determine the proximities (efficiencies, i.e., E%) of interacting proteins in sarcomeres in living cultured embryonic chicken skeletal muscle cells to draw up a FRETsome of a sarcomere in mature myofibrils. During this study, two known Z-band proteins, FATZ (also called myozenin), and telethonin, were found to be actin binding proteins. The Efficiency of the F-actin and telethonin pair was measured to be 15%, while the F-actin and FATZ pair was 18%. The known F-actin and alpha-actinin interactions resulted in an efficiency of 13%. The new actin binding properties of FATZ and telethonin were confirmed by F-actin pull down experiments. By placing a donor probe (CeFP) on one end of the FATZ molecule and the acceptor probe (YFP) on the other end of FATZ, we were able to determine that the two termini moved apart from each other as the dense bodies of the nascent myofibrils fused with one another to form the Z-bands of mature myofibrils. Three other proteins of the dense bodies, i.e., myotilin, alpha-actinin and ArgBP2 also underwent similar termini separation changes as the dense bodies transformed into Z-bands of mature myofibrils. The highest efficiencies were measured for MyBP-C interactions with myosin heavy chains (35%), myosin light chains (35%), and F-actin (31%). The establishment of a sarcomere FRETsome of these interactions in control sarcomeres in mature myofibrils in living transfected skeletal muscles establishes a foundation on which the introduction of individual mutated sarcomeric proteins involved in different muscle diseases will have on their interactions with control partner sarcomeric proteins in mature myofibrils.
ABSTRACT Dr. Thomas D. Pollard's work transformed cytoskeleton research into a quantitative, mechanistic science. Over five decades, he established core principles of actin dynamics, including filament polarity, assembly kinetics, unconventional myosins, and the molecular basis of branched networks and contractile ring formation. By integrating biochemistry, structural biology, genetics, microscopy, and modeling, his studies defined the logic of cellular architecture and motility. In this interview, Dr. Pollard reflects on key discoveries that shaped the actin field and on the role of quantitative thinking in biology. Perspectives from former trainees highlight his lasting influence as a mentor and the interdisciplinary culture that shaped generations of scientists.
Neurodegenerative diseases have largely been defined by the accumulation of misfolded and aggregated proteins, while cytoskeletal disruption has typically been treated as a secondary consequence of pathology. Here, we argue that tubulin and microtubules are active determinants of whether disease-linked proteins remain functionally engaged or enter pathological assembly pathways. Recent evidence that tubulin redirects Tau:α-synuclein condensates away from oligomerization and amyloid formation toward physiological, microtubule-association competent states supports this view. On this basis, we propose that loss of productive protein-microtubule engagement shifts intracellular binding equilibria toward disengaged protein populations, available to access pathological homo- and heterotypic interactions. These macromolecular aggregates may then further disrupt microtubule organization, exacerbating cytoskeletal failure and accelerating disease progression. This view also offers an explanation for mixed pathologies, as release from one function-related interaction network expands access to several pathogenic ones. We further consider the implications of this model for therapy and biomarker development, positioning the tubulin/microtubule system as both a targetable regulator of proteostatic fate and a biologically informative readout of early pathology. In this perspective, tubulin state is not a passive marker of damage but a determinant of neurodegenerative protein behavior.
Mutations at KIF1A residue R350 are linked to hereditary spastic paraplegia type 30 (SPG30), part of the spectrum of KIF1A-associated neurological disorders (KAND). Recent high-resolution cryo-EM structures of the KIF1A R350G and R350W mutants bound to microtubules in both nucleotide-free (apo) and AMP-PNP-bound states revealed a salt bridge between KIF1A R350 and α-tubulin E415 that forms specifically in the open motor domain conformation and is abolished by both substitutions. Single-molecule motility assays further showed that disruption of this conformation-dependent electrostatic interaction increases motor velocity while reducing processivity and microtubule affinity in the apo state. These findings identify a previously unrecognized mechanism regulating KIF1A motility and highlight how subtle changes in motor mechanochemistry can contribute to KAND pathology.
Osteocytes, the master orchestrators of bone remodeling, form an extensive network of F-actin rich dendrites that enable them to sense external mechanical signals and communicate with other cells, which are essential to maintain bone homeostasis and bone mass. Loss of the osteocyte dendrite number and connectivity and similar changes in the lacunar-canalicular pore system (LCS) surrounding the osteocyte dendrites have been reported in aged animals and humans. Increasing evidence demonstrates the close association of osteocyte dendrites/LCS alterations with many bone disorders and diseases that affect the formation, maturation, maintenance, or degeneration of osteocyte dendrites. Given their intrinsic temporal and spatial variations, phenotyping the osteocyte dendrite/LCS requires rigorous evaluation of a large number of cells with reliable methods. The goal of this manuscript is to describe several easy-to-adopt strategies of phenotyping osteocyte dendrites and high-throughput analysis methods developed in previous literature and our laboratories. Protocols will be detailed for (1) preparing various murine and human bone samples; (2) fluorescence staining of the dendrite cytoskeleton; (3) silver nitrate staining of the LCS; and (4) sample imaging and analysis including macro plugins using the free software ImageJ. The protocols provide reliable methods to phenotyping osteocytes in the context of aging, diseases, and treatments.
Proper regulation of microtubule (MT) cytoskeleton forms the basis for molecular and functional polarization of neurons. The optimal growth of mechanosensory neurons in C. elegans depends on the activity of the microtubule depolymerizing enzyme, Kinesin-13/KLP-7. Yet it's unclear how KLP-7 collaborates with other microtubule regulators in neurons for proper MT regulation. Using the ectopic growth of mechanosensory neurons in klp-7 mutants as a phenotypic handle, we characterized its genetic interactions with known microtubule stabilizers. We found that mutations in unc-33(CRMP-2), unc-44(Ankyrin), or components of the unc-14/unc-51/vab-8 pathway suppress klp-7(0) ectopic growth. Quantification of EBP-2::GFP dynamics revealed that these factors promote microtubule stability in axon-like PLM anterior neurites, suggesting an antagonistic role to KLP-7. Additionally, mec-7 (β-tubulin), unc-33, and unc-44 proteins promote the plus-end-out microtubule polarity in these neurites, and their loss in klp-7(0) mutant can reciprocally suppress the microtubule dynamics defects observed in single mutants. Notably, the simultaneous loss of klp-7 and mec-7 restored steady-state microtubule dynamics and wild-type-like PLM morphology. Together, our findings suggest that the antagonistic interplay between microtubule depolymerizing kinesin KLP-7 and microtubule-stabilizing factors, UNC-44, UNC-33, and tubulins, is necessary to maintain steady-state microtubule dynamics and plus-end-out axonal microtubule polarity in neurons.
Stick insect sperm exhibit a remarkable modification of the basal body, characterized by the expansion of the proximal region and the incorporation of amorphous, unstructured material. This atypical architecture parallels transformations observed in mammalian sperm, where the distal centriole transitions into splayed microtubules of variable length. Despite their structural irregularity, these modified centrioles enable zygotic centrosome formation, likely relying on a large permissive recruitment domain of specific proteins. Therefore, atypical centrioles may represent a sort of assembly platform, rather than providing a defined basal scaffold as observed in other systems. Basal stick insects possess canonical cylindrical centrioles, whereas higher stick insects show increasing basal body complexity. This trend mirrors mammalian sperm evolution, suggesting structural parallels of the basal bodies, presumably enhancing mechanical support to the sperm neck during flagellar motion.
The raphidophyte Chattonella marina is a harmful algal bloom (HAB) species known for its distinct diurnal vertical migration (DVM), a behavior important for its survival and bloom formation. However, the single-cell mechanisms governing this migration remain unclear. In this study, we investigated the swimming characteristics of individual C. marina cells during day (light) and night (dark) periods. We observed a strong positive correlation between the length of the propulsive anterior flagellum and the cell's swimming speed. We discovered that the length distribution of the anterior flagellum is different during the day and at night. We also found that the beat frequency of the anterior flagellum was significantly higher during the day compared to the night. This resulted in faster mean swimming speeds during the light period. To investigate the mechanism of length regulation, we tested the role of intraflagellar transport (IFT) using the IFT dynein inhibitor, ciliobrevin D. Treatment with ciliobrevin D induced a time- and concentration-dependent shortening of the anterior flagellum. This is the first pharmacological evidence to suggest that an IFT-like mechanism may actively control motile flagellar length in C. marina. These findings suggest that C. marina modulates its swimming speed through diurnal changes in both flagellar length and beat frequency, likely as an energy-saving strategy coupled to its DVM.
The role of actin and its binding proteins has been discovered in cytoskeleton remodeling as well as in Epithelial-Mesenchymal Transition (EMT) of metastatic cells and apoptosis. Even minor changes in the biomolecular structure of actin and its ABPs (by binding of ligands) can lead to drastic changes in the cytoskeleton with far reaching effects per se. Agents targeting actin can, thus, be viewed as potential anti-metastatic agents. The effect of Withania somnifera (L.) Dunal (WS) on the cytoskeleton has remained relatively unexplored. The present study highlights the interaction between 20 WS phytoconstituents and 10 selected cytoskeletal proteins in silico with a view to validate and analyze the perturbation in growth and differentiation of breast cancer cells in vitro. Pharmacokinetic analyses revealed that the majority of WS phytoconstituents exhibited no violations of Lipinski's rule-of-five parameters. Withanolides A, B, D, M and O displayed the greatest binding affinity particularly for coronin1A, vimentin, gelsolin, ezrin and F-actin. MD simulations of 100 ns revealed maximum stable interaction(s) between Coronin-Viscosalactone B (VISCB) and Vimentin-Withanolide E (WITHE). The prepared methanolic extract of WS stem (WSME), characterized using LC-MS, revealed the presence of Withaferin A (WFA). Both WSME and WFA exhibited potent cytotoxicity against breast cancer MDA-MB-231 cells. WSME increased ROS levels, arrested the cell cycle in S and G2-M phases, decreased the expression of mesenchymal markers, namely, vimentin, N-cadherin and increased levels of the epithelial marker E-cadherin in treated MDA-MB-231 cells. These findings suggest that VISCB, WITHE, and WFA have the potential to emerge as potential antimetastatic agents against breast cancer in the future.
