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Microtubules are rigid cytoskeletal filaments, and their mechanics affect cell morphology and cellular processes. For instance, microtubules for the support structures for extended morphologies, such as axons and cilia. Further, microtubules act as tension rods to pull apart chromosomes during cellular division. Unlike other cytoskeletal filaments (e.g., actin) that work as large networks, microtubules work individually or in small groups, so their individual mechanical properties are quite important to their cellular function. In this review, we explore the past work on the mechanics of individual microtubules, which have been studied for over a quarter of a century. We also present some prospective on future endeavors to determine the molecular mechanisms that control microtubule rigidity.

Actin, the primary component of the cytoskeleton, is the most studied semiflexible filament. Yet, the dynamics of actin filamentous network is still a subject of debate. Here we show that hydrodynamic interactions may significantly alter the time scale of actin network deformation. The alteration may be easily in the range of 2–20 fold depending on the structural conformations and scales of interest. We show that for a single fiber, hydrodynamic interactions between the cytoskeletal mesh-sized segments can change the net force by up to 7 folds. We also demonstrate that cytoskeletal relaxation times are underestimated if hydrodynamic interaction effects are ignored, but bending mode shapes are not appreciably influenced. Ignoring hydrodynamic interactions can result in up to 20-fold overestimation of shear loss modulus in the 2 μm range we investigated. Moreover, in agreement with experimental studies, our models explain a highly length scale dependent loss modulus. Taken together, our data suggest that including hydrodynamic interactions is key to proper modeling and analysis of actin dynamics at any scales and dimensions, and therefore must not be neglected in future models and experimental analyses of cytoskeletal dynamics.

BackgroundBone dysplasias are a large group of disorders affecting the growth and structure of the skeletal system.MethodsIn the present study, we report the clinical and molecular delineation of a new form of syndromic autosomal recessive spondylometaphyseal dysplasia (SMD) in two Emirati first cousins. They displayed postnatal growth deficiency causing profound limb shortening with proximal and distal segments involvement, narrow chest, radiological abnormalities involving the spine, pelvis and metaphyses, corneal clouding and intellectual disability. Whole genome homozygosity mapping localised the genetic cause to 11q12.1–q13.1, a region spanning 19.32 Mb with ~490 genes. Using whole exome sequencing, we identified four novel homozygous variants within the shared block of homozygosity. Pathogenic variants in genes involved in phospholipid metabolism, such as PLCB4 and PCYT1A, are known to cause bone dysplasia with or without eye anomalies, which led us to select PLCB3 as a strong candidate. This gene encodes phospholipase C β 3, an enzyme that converts phosphatidylinositol 4,5 bisphosphate (PIP2) to inositol 1,4,5 triphosphate (IP3) and diacylglycerol.ResultsThe identified variant (c.2632G>T) substitutes a serine for a highly conserved alanine within the Ha2’ element of the proximal C-terminal domain. This disrupts binding of the Ha2’ element to the catalytic core and destabilises PLCB3. Here we show that this hypomorphic variant leads to elevated levels of PIP2 in patient fibroblasts, causing disorganisation of the F-actin cytoskeleton.ConclusionsOur results connect a homozygous loss of function variant in PLCB3 with a new SMD associated with corneal dystrophy and developmental delay (SMDCD).

Neural rosettes (NPC rosettes) are radially arranged groups of cells surrounding a central lumen that arise stochastically in monolayer cultures of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPC). Since NPC rosette formation is thought to mimic cell behavior in the early neural tube, these rosettes represent important in vitro models for the study of neural tube morphogenesis. However, using current protocols, NPC rosette formation is not synchronized and results are inconsistent among different hPSC lines, hindering quantitative mechanistic analyses and challenging live cell imaging. Here, we report a rapid and robust protocol to induce rosette formation within 6 h after evenly-sized “colonies” of NPC are generated through physical cutting of uniformly polarized NESTIN+/PAX6+/PAX3+/DACH1+ NPC monolayers. These NPC rosettes show apically polarized lumens studded with primary cilia. Using this assay, we demonstrate reduced lumenal size in the absence of PODXL , an important apical determinant recently identified as a candidate gene for juvenile Parkinsonism. Interestingly, time lapse imaging reveals that, in addition to radial organization and apical lumen formation, cells within cut NPC colonies initiate rapid basally-driven spreading. Further, using chemical, genetic and biomechanical tools, we show that NPC rosette morphogenesis requires this basal spreading activity and that spreading is tightly regulated by Rho/ROCK signaling. This robust and quantitative NPC rosette platform provides a sensitive system for the further investigation of cellular and molecular mechanisms underlying NPC rosette morphogenesis.

Background and AimsPenium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division.MethodsMicrotubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy.Key ResultsThe cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division.ConclusionsThe cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre-division wall expansion and the zone where a cross wall will form during cytokinesis. This suggests that prior to the evolution of land plants, a dynamic, cortical cytoskeletal array similar to a pre-prophase band had evolved in the charophytes. However, an interesting variation on the cortical band theme is present in Penium, where two satellite microtubule bands are produced at the onset of cell expansion, each of which is destined to become an IMB in the two daughter cells after cytokinesis. These unique cytoskeletal components demonstrate the close temporal control and highly coordinated cytoskeletal dynamics of cellular development in Penium.

Giant axonal neuropathy (GAN) is a rare autosomal recessive disorder affecting both the central and peripheral nervous systems. Cytopathologically, the disorder is characterized by giant axons with derangements of cytoskeletal components. Geneticists refined the chromosomal interval containing the locus, culminating in the cloning of the defective gene, GAN. To date, many distinct mutations scattered throughout the coding region of the locus have been reported by researchers from different groups around the world. GAN encodes the protein, gigaxonin. Recently, a genetic mouse model of the disease was generated by targeted disruption of the locus. Over the years, the molecular mechanisms underlying GAN have attracted much interest. Studies have revealed that gigaxonin appears to play an important role in cytoskeletal functions and dynamics by directing ubiquitin-mediated degradations of cytoskeletal proteins. Aberrant accumulations of cytoskeletal-associated proteins caused by a defect in the ubiquitin-proteasome system (UPS) have been shown to be responsible for neurodegeneration occurring in GAN-null neurons, providing strong support for the notion that UPS plays crucial roles in cytoskeletal functions and dynamics. However, many key questions about the disease remain unanswered.

Anterior gradient 2 (AGR2) is a protein disulfide isomerase over-expressed in numerous types of cancer. Although AGR2 plays a role in ER homeostasis, its function(s) in tumorigenesis is still elusive. Here we demonstrate that AGR2 is involved in the regulation of the β-subunit of dystroglycan (β-DG), a component of the multi-protein complex linking the extracellular matrix and cytoskeletal network. In breast cancer cells, AGR2 over-expression led to the up-regulation of β-DG but not that of α-DG, while the transcript levels of these subunits were unchanged. Conversely, the reduced expression of AGR2 caused the down-regulation of β-DG. Interestingly, induced expression of AGR2 increased the degree of co-localization of AGR2 and β-DG in the cytoplasm suggesting that AGR2 facilitates the trafficking of β-DG. In addition, AGR2 over-expression caused the re-arrangement of the actin cytoskeletal network. Presumably over-expressed AGR2 up-regulates β-DG post-transcriptionally and facilitates its trafficking, which then causes re-arrangement of the cytoskeletal network, which plays a role in the adhesion and invasion of cancer cells.

Vascular walls change their dimensions and mechanical properties adaptively in response to blood pressure. Because these responses are driven by the smooth muscle cells (SMCs) in the media, a detailed understanding of the mechanical environment of the SMCs should reveal the mechanism of the adaptation. As the mechanical properties of the media are highly heterogeneous at the microscopic level, the mechanical properties of the cells should be measured directly. The tensile properties of SMCs are, thus, important to reveal the microscopic mechanical environment in vascular tissues; their tensile properties have a close correlation with the distribution and arrangement of elements of the cytoskeletal networks, such as stress fibers and microtubules. In this review, we first introduce the experimental techniques used for tensile testing and discuss the various factors affecting the tensile properties of vascular SMCs. Cytoskeletal networks are particularly important for the mechanical properties of a cell and its mechanism of mechanotransduction; thus, the mechanical properties of cytoskeletal filaments and their effects on whole-cell mechanical properties are discussed with special attention to the balance of intracellular forces among the intracellular components that determines the force applied to each element of the cytoskeletal filaments, which is the key to revealing the mechanotransduction events regulating mechanical adaptation. Lastly, we suggest future directions to connect tissue and cell mechanics and to elucidate the mechanism of mechanical adaptation, one of the key issues of cardiovascular solid biomechanics.

Force-induced deformation of tissues is transduced to the cytoskeletal (CSK) network within cells via focal adhesions. Previous studies have characterized transfer of strains of less than 15% from the substrate to the cell, using mitochondria as surrogate markers for CSK deformation. However, it is unclear if intracellular strains determined from mitochondrial displacement accurately reflect CSK network deformation. Furthermore, previous studies have not characterized substrate–CSK network strain transfer for strain magnitudes exceeding 15%, as can occur in vivo and in cell culture studies. Here, we developed and characterized a texture correlation algorithm to address the image distortion problem caused by large strain. We then used this algorithm to characterize large compressive strain (−40%) transfer from the substrate to the CSK in living cells, using fluorescently tagged actin to perform the tracking and both fluorescently tagged actin and talin to make validation measurements. With this approach, we were able to demonstrate explicitly that substrate strain transfers directly to CSK deformation in living cells undergoing large compressive deformation, and that the strain transfer ratios are independent of cell alignment. The tools and approaches developed here enable improved characterization of cell–matrix interactions under large deformation and in doing so, may reveal new insights into mechanotransduction mechanisms in such circumstances.

Microtubules are essential structures for cellular organization. They support neuronal processes and cilia, they are the scaffolds for the mitotic spindle, and they are the tracks for intracellular transport that actively organizes material and information within the cell. The mechanical properties of microtubules have been studied for almost 30 years, yet the results from different groups are startlingly disparate, ranging over an order of magnitude. Here we present results demonstrating the effects of purification, associated-protein content, age, and fluorescent labeling on the measured persistence length using the freely fluctuating filament method. We find that small percentages (<1%) of residual microtubule-associated proteins left over in the preparation can cause the persistence length to double, and that these proteins also affect the persistence length over time. Interestingly, we find that the fraction of labeled tubulin dimers does not affect the measured persistence length. Further, we have enhanced the analysis method established by previous groups. We have added a bootstrapping with resampling analysis to estimate the error in the variance data used to determine the persistence length. Thus, we are able to perform a weighted fit to the data to more accurately determine the persistence length.