Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

VEGFD signaling balances stability and activity-dependent structural plasticity of dendrites.

Mature neurons have stable dendritic architecture, which is essential for the nervous system to operate correctly. The ability to undergo structural plasticity, required to support adaptive processes like memory formation, is still present in mature neurons. It is unclear what molecular and cellular processes control this delicate balance between dendritic structural plasticity and stabilization. Failures in the preservation of optimal dendrite structure due to atrophy or maladaptive plasticity result in abnormal connectivity and are associated with various neurological diseases. Vascular endothelial growth factor D (VEGFD) is critical for the maintenance of mature dendritic trees. Here, we describe how VEGFD affects the neuronal cytoskeleton and demonstrate that VEGFD exerts its effects on dendrite stabilization by influencing the actin cortex and reducing microtubule dynamics. Further, we found that during synaptic activity-induced structural plasticity VEGFD is downregulated. Our findings revealed that VEGFD, acting on its cognate receptor VEGFR3, opposes structural changes by negatively regulating dendrite growth in cultured hippocampal neurons and in vivo in the adult mouse hippocampus with consequences on memory formation. A phosphoproteomic screening identified several regulatory proteins of the cytoskeleton modulated by VEGFD. Among the actin cortex-associated proteins, we found that VEGFD induces dephosphorylation of ezrin at tyrosine 478 via activation of the striatal-enriched protein tyrosine phosphatase (STEP). Activity-triggered structural plasticity of dendrites was impaired by expression of a phospho-deficient mutant ezrin in vitro and in vivo. Thus, VEGFD governs the equilibrium between stabilization and plasticity of dendrites by acting as a molecular brake of structural remodeling.

Length control emerges from cytoskeletal network geometry.

Many cytoskeletal networks consist of individual filaments that are organized into elaborate higher-order structures. While it is appreciated that the size and architecture of these networks are critical for their biological functions, much of the work investigating control over their assembly has focused on mechanisms that regulate the turnover of individual filaments through size-dependent feedback. Here, we propose a very different, feedback-independent mechanism to explain how yeast cells control the length of their actin cables. Our findings, supported by quantitative cell imaging and mathematical modeling, indicate that actin cable length control is an emergent property that arises from the cross-linked and bundled organization of the filaments within the cable. Using this model, we further dissect the mechanisms that allow cables to grow longer in larger cells and propose that cell length-dependent tuning of formin activity allows cells to scale cable length with cell length. This mechanism is a significant departure from prior models of cytoskeletal filament length control and presents a different paradigm to consider how cells control the size, shape, and dynamics of higher-order cytoskeletal structures.

Improvement of osteoblast adhesion, viability, and mineralization by restoring the cell cytoskeleton after bisphosphonate discontinuation in vitro.

OBJECTIVE: Bisphosphonates are prescribed to treat excessive bone resorption in patients with osteoporosis. However, its use is associated with potential adverse effects such as medication-related osteonecrosis of the jaw, prompting the introduction of the drug holiday concept in patients prior to dentoalveolar surgery. Furthermore, bisphosphonate discontinuation has been studied in vivo, in humans, and in animal models. However, it is not known whether this approach could affect bone cells in vitro. Therefore, the objective of this study was to investigate the potential effects of bisphosphonate discontinuation on pre-osteoblast and osteoblast activities in vitro. METHODOLOGY: Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations of 1, 5, and 10 µM. Alendronate was then withdrawn at different time points. The negative control consisted of untreated cells (0 µM), while the positive control consisted of cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated. RESULTS: Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 µM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with the positive control. However, these levels did not reach those of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer periods of discontinuation were more effective in restoring nodule formation than shorter periods. Addition of alendronate resulted in an increase in the percentage of dead cells, which, in turn, decreased when alendronate was discontinued. Alendronate affected the cell cytoskeleton by disassembling actin stress fibers. Cell adhesion and cell morphological parameters were also affected by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expression. CONCLUSION: Discontinuation of alendronate helps to reverse the negative effects of the drug on cell viability, cell adhesion, and mineralization by restoring the cell cytoskeleton. Our data suggest the benefits of drug holiday and/or intermittent strategies for alendronate administration at the cellular level.

Exploring the Role of Neuropeptide PACAP in Cytoskeletal Function Using Spectroscopic Methods.

The behavior and presence of actin-regulating proteins are characteristic of various clinical diseases. Changes in these proteins significantly impact the cytoskeletal and regenerative processes underlying pathological changes. Pituitary adenylate cyclase-activating polypeptide (PACAP), a cytoprotective neuropeptide abundant in the nervous system and endocrine organs, plays a key role in neuron differentiation and migration by influencing actin. This study aims to elucidate the role of PACAP as an actin-regulating polypeptide, its effect on actin filament formation, and the underlying regulatory mechanisms. We examined PACAP27, PACAP38, and PACAP6-38, measuring their binding to actin monomers via fluorescence spectroscopy and steady-state anisotropy. Functional polymerization tests were used to track changes in fluorescent intensity over time. Unlike PACAP27, PACAP38 and PACAP6-38 significantly reduced the fluorescence emission of Alexa488-labeled actin monomers and increased their anisotropy, showing nearly identical dissociation equilibrium constants. PACAP27 showed weak binding to globular actin (G-actin), while PACAP38 and PACAP6-38 exhibited robust interactions. PACAP27 did not affect actin polymerization, but PACAP38 and PACAP6-38 accelerated actin incorporation kinetics. Fluorescence quenching experiments confirmed structural changes upon PACAP binding; however, all studied PACAP fragments exhibited the same effect. Our findings indicate that PACAP38 and PACAP6-38 strongly bind to G-actin and significantly influence actin polymerization. Further studies are needed to fully understand the biological significance of these interactions.

Culturing Primary Cortical Neurons for Live-Imaging.

Primary neuronal cultures allow for in vitro analysis of early developmental processes such as axon pathfinding and growth dynamics. When coupled with methods to visualize and measure microtubule dynamics, this methodology enables an inside look at how the cytoskeleton changes in response to extracellular signaling cues. Here, we describe the culturing conditions and tools required to extract primary cortical neurons from postnatal mouse brains and visualize cytoskeletal components.

Mechanically induced topological transition of spectrin regulates its distribution in the mammalian cell cortex.

The cell cortex is a dynamic assembly formed by the plasma membrane and underlying cytoskeleton. As the main determinant of cell shape, the cortex ensures its integrity during passive and active deformations by adapting cytoskeleton topologies through yet poorly understood mechanisms. The spectrin meshwork ensures such adaptation in erythrocytes and neurons by adopting different organizations. Erythrocytes rely on triangular-like lattices of spectrin tetramers, whereas in neurons they are organized in parallel, periodic arrays. Since spectrin is ubiquitously expressed, we exploited Expansion Microscopy to discover that, in fibroblasts, distinct meshwork densities co-exist. Through biophysical measurements and computational modeling, we show that the non-polarized spectrin meshwork, with the intervention of actomyosin, can dynamically transition into polarized clusters fenced by actin stress fibers that resemble periodic arrays as found in neurons. Clusters experience lower mechanical stress and turnover, despite displaying an extension close to the tetramer contour length. Our study sheds light on the adaptive properties of spectrin, which participates in the protection of the cell cortex by varying its densities in response to key mechanical features.

Microscale geometrical modulation of PIEZO1 mediated mechanosensing through cytoskeletal redistribution.

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1's activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.

Visualizing the Cell-Matrix Interactions and Cytoskeleton of Disseminated Tumor Cells.

Tumor cells often leave the primary tumor mass and get settled in a foreign tissue years before the development of overt metastases, exhibiting the highly inefficient nature of metastatic colony formation. In fact, the tumor cells that disseminate into distant organs and subsequently invade the parenchyma of these organs rarely proceed to found actively growing metastatic colonies. Instead, the majority of these tumor cells undergo prolonged proliferative arrest unless they are swiftly eliminated by the immune system. Together, these observations indicate that the proliferative capacity of the disseminated tumor cells (DTCs) serves as a key determinant of the efficiency of metastasis, highlighting the need to better understand the mechanism governing the proliferation of these cells. Recent studies are unveiling the importance of the interactions between DTCs and the microenvironment of the host tissue in regulating the proliferation of DTCs. However, the details of such interactions remain to be fully delineated. Here I describe the methods for visualizing and analyzing the interactions between DTCs and the extracellular matrix (ECM) components of the host tissue as well as the cytoskeleton of the DTCs that support these interactions. The methods described here will facilitate the study of how DTCs interact with the ECM of their host tissue, which will be crucial for elucidating the mechanism that underlies the regulation of DTC proliferation by the DTC-ECM interactions.

Derivation and simulation of a computational model of active cell populations: How overlap avoidance, deformability, cell-cell junctions and cytoskeletal forces affect alignment.

Collective alignment of cell populations is a commonly observed phenomena in biology. An important example are aligning fibroblasts in healthy or scar tissue. In this work we derive and simulate a mechanistic agent-based model of the collective behaviour of actively moving and interacting cells, with a focus on understanding collective alignment. The derivation strategy is based on energy minimisation. The model ingredients are motivated by data on the behaviour of different populations of aligning fibroblasts and include: Self-propulsion, overlap avoidance, deformability, cell-cell junctions and cytoskeletal forces. We find that there is an optimal ratio of self-propulsion speed and overlap avoidance that maximises collective alignment. Further we find that deformability aids alignment, and that cell-cell junctions by themselves hinder alignment. However, if cytoskeletal forces are transmitted via cell-cell junctions we observe strong collective alignment over large spatial scales.

Cytoskeleton-associated gelsolin responds to the midgut distention process in saline meal-fed Aedes aegypti and affects arbovirus dissemination from the midgut.

The mosquito, Aedes aegypti, is the principal vector for several arboviruses. The mosquito midgut is the initial tissue that gets infected with an arbovirus acquired along with a blood meal from a vertebrate host. Blood meal ingestion leads to midgut tissue distention thereby increasing the pore size of the surrounding basal lamina. This allows newly synthesized virions to exit the midgut by traversing the distended basal lamina to infect secondary tissues of the mosquito. We conducted a quantitative label-free proteomic time course analysis with saline meal-fed Ae. aegypti females to identify host factors involved in midgut tissue distention. Around 2000 proteins were detected during each of the seven sampling time points and 164 of those were uniquely expressed. Forty-five of 97 differentially expressed proteins were upregulated during the 96-h time course and most of those were involved in cytoskeleton modulation, metabolic activity, and vesicle/vacuole formation. The F-actin-modulating Ae. aegypti (Aa)-gelsolin was selected for further functional studies. Stable knockout of Aa-gelsolin resulted in a mosquito line, which showed distorted actin filaments in midgut-associated tissues likely due to diminished F-actin processing by gelsolin. Zika virus dissemination from the midgut of these mosquitoes was diminished and delayed. The loss of Aa-gelsolin function was associated with an increased induction of apoptosis in midgut tissue indicating an involvement of Aa-gelsolin in apoptotic signaling in mosquitoes. Here, we used proteomics to discover a novel host factor, Aa-gelsolin, which affects the midgut escape barrier for arboviruses in mosquitoes and apoptotic signaling in the midgut.

[EMP3 inhibits the formation of heterotypic cell-in-cell structures in hepatocellular carcinoma].

Heterotypic cell-in-cell (heCIC) structures represent a unique intercellular interaction where tumor cells internalize immune cells to enhance the killing efficiency of immune cells. However, the mechanism of heCIC structure formation remains to be fully elucidated. In this study, we explored the role of epithelial membrane protein 3 (EMP3), a PMP-22/EMP/MP20 protein family member highly expressed in the patients with hepatocellular carcinoma and poor prognosis, in the formation of the heCIC structure formed by natural killer cells and hepatocellular carcinoma cells. The analysis of monoclonal hepatocellular carcinoma cell lines revealed that EMP3 presented low expression in the cells with high capability to form heCIC structure and high expression in those with low capability. Knocking down the expression of EMP3 by gene editing promoted the formation of heCIC structures, while overexpression of EMP3 significantly inhibited this process. Additionally, the expression of factors involved in the heCIC structure formation suggested that EMP3 inhibited the formation of heCIC structures by modulating the adhesion ability and cytoskeleton of tumor cells. The findings lay a foundation for enhancing the heCIC-mediated tumor immunotherapy by targeting EMP3.

Mechanical regulation of lymphocyte activation and function.

Mechanosensing, or how cells sense and respond to the physical environment, is crucial for many aspects of biological function, ranging from cell movement during development to cancer metastasis, the immune response and gene expression driving cell fate determination. Relevant physical stimuli include the stiffness of the extracellular matrix, contractile forces, shear flows in blood vessels, complex topography of the cellular microenvironment and membrane protein mobility. Although mechanosensing has been more widely studied in non-immune cells, it has become increasingly clear that physical cues profoundly affect the signaling function of cells of the immune system. In this Review, we summarize recent studies on mechanical regulation of immune cells, specifically lymphocytes, and explore how the force-generating cytoskeletal machinery might mediate mechanosensing. We discuss general principles governing mechanical regulation of lymphocyte function, spanning from the molecular scale of receptor activation to cellular responses to mechanical stimuli.

Cytoplasmic stirring by active carpets.

Large cells often rely on cytoplasmic flows for intracellular transport, maintaining homeostasis, and positioning cellular components. Understanding the mechanisms of these flows is essential for gaining insights into cell function, developmental processes, and evolutionary adaptability. Here, we focus on a class of self-organized cytoplasmic stirring mechanisms that result from fluid-structure interactions between cytoskeletal elements at the cell cortex. Drawing inspiration from streaming flows in late-stage fruit fly oocytes, we propose an analytically tractable active carpet theory. This model deciphers the origins and three-dimensional spatiotemporal organization of such flows. Through a combination of simulations and weakly nonlinear theory, we establish the pathway of the streaming flow to its global attractor: a cell-spanning vortical twister. Our study reveals the inherent symmetries of this emergent flow, its low-dimensional structure, and illustrates how complex fluid-structure interaction aligns with classical solutions in Stokes flow. This framework can be easily adapted to elucidate a broad spectrum of self-organized, cortex-driven intracellular flows.

Multi-domain interaction mediated strength-building in human α-actinin dimers unveiled by direct single-molecule quantification.

α-Actinins play crucial roles in cytoskeletal mechanobiology by acting as force-bearing structural modules that orchestrate and sustain the cytoskeletal framework, serving as pivotal hubs for diverse mechanosensing proteins. The mechanical stability of α-actinin dimer, a determinant of its functional state, remains largely unexplored. Here, we directly quantify the force-dependent lifetimes of homo- and hetero-dimers of human α-actinins, revealing an ultra-high mechanical stability of the dimers associated with > 100 seconds lifetime within 40 pN forces under shear-stretching geometry. Intriguingly, we uncover that the strong dimer stability is arisen from much weaker sub-domain pair interactions, suggesting the existence of distinct dimerized functional states of the dimer, spanning a spectrum of mechanical stability, with the spectrin repeats (SRs) in folded or unfolded conformation. In essence, our study supports a potent mechanism for building strength in biomolecular dimers through weak, multiple sub-domain interactions, and illuminates multifaceted roles of α-actinin dimers in cytoskeletal mechanics and mechanotransduction.

Perturbed N-glycosylation of Halobacterium salinarum archaellum filaments leads to filament bundling and compromised cell motility.

The swimming device of archaea-the archaellum-presents asparagine (N)-linked glycans. While N-glycosylation serves numerous roles in archaea, including enabling their survival in extreme environments, how this post-translational modification contributes to cell motility remains under-explored. Here, we report the cryo-EM structure of archaellum filaments from the haloarchaeon Halobacterium salinarum, where archaellins, the building blocks of the archaellum, are N-glycosylated, and the N-glycosylation pathway is well-resolved. We further determined structures of archaellum filaments from two N-glycosylation mutant strains that generate truncated glycans and analyzed their motility. While cells from the parent strain exhibited unidirectional motility, the N-glycosylation mutant strain cells swam in ever-changing directions within a limited area. Although these mutant strain cells presented archaellum filaments that were highly similar in architecture to those of the parent strain, N-linked glycan truncation greatly affected interactions between archaellum filaments, leading to dramatic clustering of both isolated and cell-attached filaments. We propose that the N-linked tetrasaccharides decorating archaellins act as physical spacers that minimize the archaellum filament aggregation that limits cell motility.

Organogenesis: How active forces maintain integrity of migrating cells under pressure.

Cells experience dynamic internal and external forces during animal development. Two new studies reveal critical and unexpected roles for cytoskeletal regulators and nuclear positioning in maintaining the physical integrity of migrating leader cells during Caenorhabditis elegans organogenesis.

A circle of life: platelet and megakaryocyte cytoskeleton dynamics in health and disease.

Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.