Tubulin superfamily (TSF) proteins are widespread and known for multifaceted roles as cytoskeletal proteins underpinning many basic cellular functions including morphogenesis, division, and motility. In eukaryotes, tubulin assembles into microtubules, a major component of the dynamic cytoskeletal network of fibres, whereas the bacterial homolog FtsZ assembles the division ring at midcell. Functions of the lesser-known archaeal TSF proteins are beginning to be identified, and show surprising diversity, including homologs of tubulin and FtsZ, and a third archaea-specific family, CetZ, implicated in the regulation of cell shape and possibly other unknown functions. In this study, we defined sequence and structural characteristics of the CetZ family and CetZ1 and CetZ2 subfamilies, identified CetZ groups and diversity amongst archaea, and identified potential functional relationships through analysis of the genomic neighbourhoods of cetZ genes. At least three subfamilies of orthologous CetZ proteins were identified in the archaeal class Halobacteria, including CetZ1 and CetZ2 and a novel uncharacterized subfamily. CetZ1 and CetZ2 were correlated to one another and to cell shape and motility phenotypes across diverse Halobacteria. Amongst other known CetZ clusters in orders Archaeoglobales, Methanomicrobiales, Methanosarcinales, and Thermococcales, an additional uncharacterized group from Archaeoglobales and Methanomicrobiales affiliated strongly with Halobacteria CetZs, suggesting they originated via horizontal transfer. Subgroups of Halobacteria CetZ2 and Thermococcales CetZ genes were found adjacent to different type IV pili regulons, suggesting a potential utilization of CetZs by type IV systems. More broadly conserved cetZ gene neighbourhoods included nucleotide and cofactor biosynthesis (e.g., F420) and predicted cell surface sugar epimerase genes. The findings imply that CetZ subfamilies are involved in multiple functions linked to the cell surface, biosynthesis and motility.

The actin cytoskeleton is crucially important to maintenance of cellular structure, cell motility and endocytosis. Accordingly, bacterial pathogens often co-opt the actin-restructuring machinery of host cells to access or create a favorable environment for their own replication. The obligate intracellular organism Chlamydia trachomatis and related species exemplify this dynamic: by inducing actin polymerization at the site of pathogen-host attachment, Chlamydiae induce their own uptake by the typically non-phagocytic epithelium they infect. The interaction of chlamydial adhesins with host surface receptors has been implicated in this effect, as has the activity of the chlamydial effector TarP (translocated actin recruitment protein). Following invasion, C. trachomatis dynamically assembles and maintains an actin-rich cage around the pathogen’s membrane-bound replicative niche, the chlamydial inclusion. Through further induction of actin polymerization and modulation of the actin-crosslinking protein myosin II, C. trachomatis promotes egress from the host via extrusion of the inclusion. In this review, we present the experimental findings that inform our understanding of actin-dependent chlamydial pathogenesis, discuss lingering questions, and identify potential avenues of future study.

Nanoparticles (NPs) are commonly used in healthcare and nano therapy, but their toxicity at high concentrations is well-known. Recent research has shown that NPs can also cause toxicity at low concentrations, disrupting various cellular functions and leading to altered mechanobiological behavior. While researchers have used different methods to investigate the effects of NPs on cells, including gene expression and cell adhesion assays, the use of mechanobiological tools in this context has been underutilized. This review emphasizes the importance of further exploring the mechanobiological effects of NPs, which could reveal valuable insights into the mechanisms behind NP toxicity. Such investigations could aid in developing new strategies to mitigate NP toxicity and improve their safety for biomedical applications. Moreover, understanding how NPs affect cell cytoskeletal functions through mechanobiology could have significant implications, including the development of innovative drug delivery systems and tissue engineering techniques. In summary, this review highlights the significance of incorporating mechanobiology into the study of NP toxicity and demonstrates the potential of this interdisciplinary field to advance our knowledge and practical use of NPs.

Trypanosome brucei, the causative agent of African sleeping sickness, harbours a highly ordered, subpellicular microtubule cytoskeleton that defines many aspects of morphology, motility and virulence. This array of microtubules is associated with a large number of proteins involved in its regulation. Employing proximity-dependent biotinylation assay (BioID) using the well characterized cytoskeleton-associated protein CAP5.5 as a probe we identified CAP50 (Tb927.11.2610). This protein colocalizes with the subpellicular cytoskeleton microtubules but not with the flagellum. Depletion by RNAi results in defects in cytokinesis, morphology and partial disorganization of microtubule arrays. Published proteomics data indicate a possible association of CAP50 with two other, yet uncharacterized, cytoskeletal proteins, CAP52 (Tb927.6.5070) and CAP42 (Tb927.4.1300), which were therefore included in our analysis. We show that their depletion causes phenotypes similar to those described for CAP50 and that they are essential for cellular integrity.

The nuclear architecture describes the organization of the various compartments in the nucleus of eukaryotic cells, where a plethora of processes such as nucleocytoplasmic transport, gene expression, and assembly of ribosomal subunits occur in a dynamic manner. During the different phases of the cell cycle, in post-mitotic cells and after oncogenic transformation, rearrangements of the nuclear architecture take place, and, among other things, these alterations result in reorganization of the chromatin and changes in gene expression. A member of the tubulin family, tubulin, was first identified as part of a multiprotein complex that allows nucleation of microtubules. However, more than a decade ago, γ tubulin was also characterized as a nuclear protein that modulates several crucial processes that affect the architecture of the nucleus. This review presents the latest knowledge regarding changes that arise in the nuclear architecture of healthy cells and under pathological conditions and, more specifically, considers the particular involvement of tubulin in the modulation of the biology of the nuclear compartment.

The highly specialized structure and function of neurons depend on a sophisticated organization of the cytoskeleton, which supports a similarly sophisticated system to traffic organelles and cargo vesicles. Mitochondria sustain crucial functions by providing energy and buffering calcium where is needed. Accordingly, the distribution of mitochondria is not even in neurons and is regulated by a dynamic balance between active transport and stable docking events. This system is finely tuned to respond to changes in environmental conditions and neuronal activity. In this review, we summarize the mechanisms by which mitochondria are selectively transported in different compartments taking into account the structure of the cytoskeleton, the molecular motors and the metabolism of neurons. Remarkably, the motor proteins driving the mitochondrial transport in axons have been shown to mediate also their transfer between cells. This so-named intercellular transport of mitochondria is opening new exciting perspectives in the treatment of multiple diseases.

Vimentin has been reported to play diverse roles in cell processes such as spreading, migration, and cell-matrix adhesion. Here, we use vimentin knock out cells (Vim KO) to assess how vimentin impacts cell spreading, morphology, and myofibroblast transformation of human corneal fibro-blasts. Overall, loss of vimentin did not significantly impact corneal fibroblast spreading, as indicated by Vim KO morphology and mechanical activity (traction force). However, we found that Vim KO cells had reduced elongation in response to PDGF as compared to controls. Furthermore, absence of vimentin did not completely block TGFβ induced myofibroblast transformation, since αSMA protein expression was detected in Vim KO cells. Nonetheless, the degree of transformation and amount of αSMA protein was reduced as compared to control cells. Proteomics showed that Vim KO cells cultured in TGFβ had a similar pattern of protein expression as controls. One exception included periostin, an ECM protein associated with wound healing and fibrosis in other cell types, which was highly expressed only in Vim KO cells. We also demonstrate for the first time that LRRC15, a protein previously associated with myofibroblast transformation of cancer-associated fibroblasts, is also expressed by corneal myofibroblasts. Overall, our data shows that vimentin has a limited, yet measurable impact on corneal fibroblast spreading and myofibroblast transformation. We also identified novel proteins that may regulate corneal myofibroblast transformation in the presence and/or absence of vimentin.

The relationship between Parkinson's disease (PD), the second most common neurodegenerative disease after Alzheimer's disease, and palmitoylation, a post-translational lipid modification, is not well understood. In this study, we have analyzed the differential palmitome present in the cerebral cortex of PD patients compared to controls (n=4 per group) to better understand the role of protein palmitoylation in PD and the pathways altered in this disease. Data-mining of the cortical palmitome from PD patients and controls has allowed to: i) detect a set of 150 proteins with altered palmitoylation in PD subjects in comparison with controls, ii) describe the biological pathways and targets predicted to be altered by these palmitoylation changes, and iii) depict the overlap between the differential palmitome identified in our study with protein interactomes of the PD-linked proteins α-synuclein, LRRK2, DJ-1, PINK1, GBA and UCHL1. In summary, we have partially characterized the altered palmitome in the cortex of PD patients which is predicted to impact cytoskeleton, mitochondrial and fibrinogen functions, as well as cell survival. Our study points out that protein palmitoylation could have a role in the pathophysiology of PD, and that comprehensive palmitoyl-proteomics offers a powerful approach for elucidating novel cellular pathways modulated in this neurodegenerative disease.

Mechanical properties of tumor cytoskeleton are extremely vital for any phases of cancer, especially in tumor invasion and metastasis. However, in current category of anticancer drugs, the cytoskeleton-targeting drugs are limited and its role in tumor progression is unclear. Here, we present the mechanical characteristics of tumor stiffness are tightly regulated by cancer cytoskeleton including actin filaments and microtubule during tumor initiation, growth and metastasis, and review the natural drugs that target cancer cytoskeleton. We define cytoskeleton dynamics as target mechanisms for anticancer drug, and summary the plant, microbial and marine sources of natural products. Furthermore, the material approaches to active cancer mechanics are supplied in this review. We aim to promote the development of anticancer drugs that target tumor mechanics by using those material approaches in future and find its pharmacological application.

Charcot-Marie-Tooth (CMT) disease is the most commonly inherited neurological disorder, defined by progressive deterioration of the peripheral nerves. Clinical manifestations of CMT mutations are typically limited to peripheral neurons, the longest cells in the body. Currently, mutations in at least 80 different genes are associated with CMT and new mutations are regularly being discovered. A large portion of the proteins mutated in axonal CMT have documented roles in mitochondrial mobility. This suggests that trafficking defects may be a common underlying mechanism in CMT. This review will focus on the potential role of altered mitochondrial mobility in the pathogenesis of axonal CMT, highlighting the challenges and opportunities to this “impaired mobility” model of the disease.

Purpose: This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor, in a porcine ex vivo pigmentary glaucoma model. Methods: Twenty-eight porcine anterior chambers were perfused with medium supplemented with 1.67 × 10⁷ pigment particles/mL for 48 hours before treatment with RKI-1447 (n = 16) or vehicle control (n = 12). Intraocular pressure (IOP) was recorded and outflow facility was calculated. Primary trabecular meshwork cells were exposed to RKI-1447 or vehicle control; effects on the cytoskeleton, motility, and phagocytosis were evaluated. Result: Compared to baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 mmHg to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 hours (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 caused no apparent histological changes in the micro- or macroscopic TM appearance. RKI-1447-treated primary TM cells showed significant disruption of the actin cytoskeleton both in the presence and absence of pigment (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI-1447 reversed. Conclusion: RKI-1447 significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it useful for the treatment of secondary glaucomas with an increased phagocytic load.

Background: Epithelial-mesenchymal transition (EMT) is a biological process where epithelial cells lose their adhesive properties and gain invasive, metastatic, and mesenchymal properties. Maintaining the balance between epithelial and mesenchymal stage is essential for tissue homeo-stasis. Many of the genes promoting mesenchymal transformation has been identified; however, our understanding of the genes responsible for maintaining the epithelial phenotype is limited. Our objective was to identify genes responsible for maintaining the epithelial phenotype and in-hibiting EMT. Methods: RNA seq was performed using an vitro model of EMT. CTGF expres-sion was determined by qPCR and Western blot analysis. Knockout of CTGF was done using the CTGF sgRNA CRISPR/CAS9. Tumorigenic potential was determined using NCG mice. Results: Knocked-out of CTGF in epithelial ovarian cancer cells leads to the acquisition of functional characteristics associated with the mesenchymal phenotype such as Anoikis resistance, cytoskel-eton remodeling, increased cell stiffness, and acquisition of invasion and tumorigenic capacity. Conclusions: We identified CTGF is an important regulator of the epithelial phenotype, and its loss is associated with early cellular modifications required for EMT. We describe a novel role for CTGF, regulating cytoskeleton and the extracellular matrix interactions necessary for conserva-tion of epithelial structure and function. These findings provide a new window to understand the early stages on mesenchymal transformation

Hereditary Parkinson’s disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCAA53T in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was detected also for other microtubule and microfilament factors, namely MAP2S1801, MARK1S394, MAP1AT1794, KIF1AS1537, 4.1NS541, 4.1GS86 and ADD2S528. While the MAP1B heavy chain supports regeneration and growth cones, its light-chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2S299, VPS13DS2429 and VPS13CS2480 in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in D. melanogaster that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly, ANK2 gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.

Rotavirus (RV) replicates within viroplasms, membrane-less electron-dense globular cytosolic inclusions with liquid-liquid phase properties. In these structures occur the virus transcription, replication, and packaging of the virus genome in newly assembled double-layered particles. The viroplasms are composed of virus proteins (NSP2, NSP5, NSP4, VP1, VP2, VP3, and VP6), single and double-stranded virus RNAs, and host components such as microtubules, perilipin-1 and chaperonins. The formation, coalescence, maintenance, and perinuclear localization of viroplasms rely on their association with the cytoskeleton. A stabilized microtubule network involving microtubules and kinesin Eg5 and dynein molecular motors is associated with NSP5, NSP2, and VP2, facilitating dynamic processes such as viroplasm coalescence and perinuclear localization. Key post-translation modifications, particularly phosphorylation events of RV proteins NSP5 and NSP2, play pivotal roles in orchestrating these interactions. Actin filaments also contribute, triggering the formation of the viroplasms through the association of soluble cytosolic VP4 with actin and the molecular motor myosin. This review explores the evolving understanding of RV replication, emphasizing the host requirements essential for viroplasm formation and highlighting their dynamic interplay within the host cell.

Elevation of the intermediate amino acid metabolite Homocysteine (Hcy) causes Hyperhomocysteinemia (HHcy), a metabolic disorder frequently associated with mutations in the methionine-cysteine metabolic cycle as well as with nutritional deficiency and aging. Previous literature suggest that HHcy is a strong risk factor for cardiovascular diseases. Severe HHcy is well established to correlate with vascular pathologies primarily via endothelial cell death. Though moderate HHcy is more prevalent and associated with an increased risk of cardiovascular abnormalities in later part of life, its precise role in endothelial physiology is largely unknown. In this study, we report that moderate elevation of Hcy causes endothelial dysfunction through impairment of their migration and proliferation. We established that unlike severe elevation of Hcy, moderate HHcy is not associated with suppressed endothelial VEGF/VEGFR signaling and oxidative stress induction. We further showed that moderate HHcy induces a sub-lethal ER stress that causes defective endothelial migration through abnormal actin cytoskeletal remodeling. We also found that sub-lethal increase of Hcy causes endothelial proliferation defect by suppressing mitochondrial respiration and concomitantly increases glycolysis to compensate the consequential ATP loss and maintain overall energy homeostasis. Finally, analyzing an available microarray dataset, we confirmed that these hallmarks of moderate HHcy are conserved in adult endothelial cells as well. Thus, we identified adaptive UPR and metabolic rewiring as two key mechanistic signatures in moderate HHcy-associated endothelial dysfunction. As HHcy is clinically associated with enhanced vascular inflammation and hypercoagulability, identifying these mechanistic pathways may serve as future targets to regulate endothelial function and health.

Desmin is the major intermediate filament protein of all three muscle cell types and connects different cell organelles and multi-protein complexes like the cardiac desmosomes. Several pathogenic mutations in the DES gene cause different skeletal and cardiac myopathies. However, the significance of the majority of DES missense variants is currently unknown since functional data are lacking. To determine whether desmin missense mutations within the highly conserved 1A coil domain cause a filament assembly defect, we generated a set of variants with unknown significance and analyzed systematically the filament assembly in transfected SW13 and H9c2 cells using confocal microscopy. We found that mutations in the N-terminal part of the 1A coil domain affect the filament assembly leading to the cytoplasmic desmin aggregation. In contrast, mutant desmin in the C-terminal part of the 1A coil domain form filamentous structures comparable to wild-type desmin. Our findings suggest that the N-terminal part of the 1A coil domain is a hot spot for pathogenic desmin mutations, which affect the desmin filament assembly leading in consequence to skeletal and/or cardiac myopathies. This study may have relevance for the genetic counselling of patients carrying variants in the 1A coil domain of the DES gene.

One of the major unsolved mysteries of biological science concerns the question of where and in what form information is stored in the brain. I propose that memory is stored in the brain in a mechanically encoded binary format written into the conformations of proteins found in the cell-extracellular matrix adhesions that organise each and every synapse. The MeshCODE framework outlined here represents a unifying theory of data storage in animals, providing read-write storage of both dynamic and persistent information in a binary format. Mechanosensitive proteins that contain force-dependent switches can store information persistently, which can be written or updated using small changes in mechanical force. These mechanosensitive proteins, such as talin, scaffold each synapse, creating a meshwork of switches that together form a code, the so-called MeshCODE. Large signalling complexes assemble on these scaffolds as a function of the switch patterns and these complexes would both stabilise the patterns and coordinate synaptic regulators to dynamically tune synaptic activity. Synaptic transmission and action potential spike trains would operate the cytoskeletal machinery to write and update the synaptic MeshCODEs, thereby propagating this coding throughout the organism. Based on established biophysical principles, such a mechanical basis for memory would provide a physical location for data storage in the brain, with the binary patterns, encoded in the information-storing mechanosensitive molecules in the synaptic scaffolds, and the complexes that form on them, representing the physical location of engrams. Furthermore, the conversion and storage of sensory and temporal inputs into a binary format would constitute an addressable read-write memory system, supporting the view of the mind as an organic supercomputer.

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. We present here a shot-gun differential proteomic analysis on roots of two pldα1 mutants compared to the Col-0 wild type. Our data suggest new roles of PLDα1 in endomembrane transport, mitochondrial protein import and protein quality control and glucosinolate biosynthesis. Thus, we identified proteins involved in endocytosis, endoplasmic reticulum-Golgi transport and attachment sites of endoplasmic reticulum and plasma membrane (V-type proton ATPases, protein transport protein SEC13 homolog A, vesicle-associated protein 1-2, vacuolar protein sorting-associated protein 29, syntaxin-32, all upregulated in the mutants), mitochondrial import and electron transport chain (mitochondrial import inner membrane translocase subunits TIM23-2 and TIM13, mitochondrial NADH dehydrogenases, ATP synthases, cytochrome c oxidase subunit 6b-1, ADP,ATP carrier protein 2, downregulated in the mutants) and glucosinolate biosynthesis (3-isopropylmalate dehydrogenases 1, 2 and 3, methylthioalkylmalate synthase 1, cytochrome P450 83B1, Glutathione S-transferase F9, indole glucosinolate O-methyltransferase 1, adenylyl-sulfate kinase 1, all upregulated in mutants). Our results suggest broader biological roles of PLDα1 as anticipated so far.

Microcystins (MCs) are cyanobacterial toxins and potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A), which are involved in plant cytoskeleton (microtubules and F-actin) organization. Therefore, studies on the toxicity of cyanobacterial products on plant cells have so far being focused on MCs. In this study, we investigated the effects of extracts from 16 (4 MC-producing and 12 non-MC-producing) cyanobacterial strains from several habitats, on various enzymes (PP1, trypsin, elastase), on the plant cytoskeleton and H2O2 levels in Oryza sativa (rice) root cells. Seedling roots were treated for various time periods (1, 12 and 24h) with aqueous cyanobacterial extracts and underwent either immunostaining for α-tubulin or staining of F-actin with fluorescent phalloidin. DCF-DA staining was performed for H2O2 imaging. The enzyme assays confirmed the bioactivity of the extracts of not only MC-rich (MC+), but also MC-devoid (MC-) extracts, which induced major time-dependent alterations on both components of the plant cytoskeleton. These findings suggest that a broad spectrum of bioactive cyanobacterial compounds, apart from MCs or other known cyanotoxins (such as cylindrospermopsin), can affect plants by disrupting the cytoskeleton.

The uneven immunogenicity of the attenuated tetravalent dengue vaccine has created the diffi-culty to achieve a balanced protection against all four serotypes of dengue virus (DENV). To overcome this problem, non-replicative-based vaccines have come into focus as their immuno-genicity is adjustable. This approach is excellent for multivalent vaccines but commonly faces the issue of low immunogenicity. In this present study, we developed a non-replicating dengue vaccine composing of UV-inactivated dengue-2 virus (UV-DENV2) and DENV-2 NS1-279 protein encapsidated within nanoparticles. This vaccine candidate was administered in the presence of BCG cell wall cytoskeleton (BCG-CWS) as an adjuvant. We revealed, here, that encapsidated immunogens with BCG-CWS exerted potent activities on both B and T cells and elicited Th-1/Th-2 responses in mice. This was evidenced by strong neutralizing antibody activity with an unde-tectable antibody enhancing activity in a group of mice receiving encapsidated immunogens with BCG-CWS. Importantly, BCG-CWS significantly augmented antibody-mediated comple-ment-fixing activity, strongly stimulated the antigen-specific polyfunctional T cell responses as well as activated mixed Th-1/Th-2 responses specific to DENV2- and NS1-279 antigens. In conclu-sion, BCG-CWS potently adjuvanted the inactivated DENV-2 and subunit DENV immunogens. The mechanism of adjuvanticity remains unclear. This study revealed the potential use of BCG-CWS in vaccine development.

Citrus greening disease or huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) limits the citrus production worldwide. CLas is transmitted by the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae) in a persistent-propagative manner. Application of insecticides to manage the psyllid vectors and disease is the most common practice. Understanding the molecular interaction between CLas and ACP and interrupting the interrelationship can provide an alternative to insecticides for managing citrus greening disease. Transcriptome analysis of ACP in response to CLas showed differential expression of 3911 genes (2196 up-regulated, and 1715 down-regulated) including the key genes of ACP involved in cytoskeleton synthesis and nutrition-related proteins. Majority of the differentially expressed genes were categorized under molecular function followed by cellular components and biological processes. KEGG pathway analysis showed differential regulation of carbohydrate, nucleotide and energy metabolic pathways, the endocytotic pathway and the defense-related pathways. Differential regulation of genes associated with the key pathways might favors CLas to become systemic and propagate in its insect vector. The study provides an understanding of genes involved in circulation of CLas in ACP. The candidate genes involved in key physiological processes and CLas transmission by ACP would be potential targets for sustainable management of ACP and CLas.

Pigment dispersion can lead to pigmentary glaucoma, a poorly understood condition of younger myopic eyes with fluctuating high intraocular pressure. It has been difficult to investigate its pathogenesis without a model similar to human eyes in size and behavior. Here we present a porcine ex vivo model that recreates several features of pigmentary glaucoma, including intraocular hypertension, accumulation of pigment in the trabecular meshwork, and declining phagocytosis. We found that trabecular meshwork cells regulate outflow, form actin stress fibers, and have a decreased phagocytic activity. Gene expression microarrays and a pathway analysis of TM monolayers as well as ex vivo anterior segment perfusion cultures indicated that RhoA plays a central role in regulating the cytoskeleton, motility, and phagocytosis in the trabecular meshwork, providing new insights and targets to investigate in pigmentary glaucoma.

Pandemics caused by viruses have threatened lives of thousands of people. Viral infection is a complex and diverse process, and substantial studies have been complemented in understanding the biochemical and molecular interactions between viruses and hosts. However, the physical microenvironment where infections implement is often less carefully considered, and the role of mechanobiology in viral infection remains elusive. Mechanobiology focuses on sensation, transduction and response to intracellular and extracellular physical factors by tissues, cells and extracellular matrix. The intracellular cytoskeleton and mechanosensors have been proved to be extensively involved in virus life cycle. Furthermore, innovative methods in vivo and in vitro are being utilized to elucidate how extracellular factors including stiffness, forces and topography in regulating viral infection. Our current review covers how physical factors from different sources coordinate virus infection. We further discuss how this knowledge can be harnessed in future research on cross-fields of mechanobiology and virology.

Downregulated microRNA-142-3p signaling contributes to the pathogenesis of endometriosis [1] [2], an invasive disease where the lining of the uterus grows at ectopic locations, by yet incompletely understood mechanisms. Using bioinformatics and in vitro assays, this study identifies cytoskeletal regulation and integrin signaling as two relevant categories of miR-142-3p targets. qPCR revealed that miR-142-3p upregulation in St-T1b cells downregulates ROCK2, CFL2, RAC1, WASL and ITGAV. qPCR and Western-blotting showed miR-142-3p effect on WASL and ITGAV was significant also in primary endometriotic stroma cells. Luciferase reporter assays in ST-T1b cells then confirmed direct regulation of ITGAV and WASL. On the functional side, miR-142-3p upregulation significantly reduced ST-T1b cell size, the size of vinculin plaques, migration through fibronectin-coated transwell filters and the ability of ST-T1b and primary endometriotic stroma cells to contract collagen I gels. These results suggest that miR-142-3p has a strong mechanoregulatory effect on endometrial stroma cells and its external administration reduces the invasive endometrial phenotype. Within the limits of an in vitro investigation, our study provides new mechanistic insights into the pathogenesis of endometriosis and provides a perspective for the development of miR-142-3p based drugs for inhibiting invasive growth of endometriotic cells.

Purpose: To investigate the effect of pigment dispersion on trabecular meshwork (TM) cells. Methods: Porcine TM cells from ab interno trabeculectomy specimens were exposed to pigment dispersion, then analyzed for changes in morphology, immunostaining, and ultrastructure. Their abilities to phagocytose, migrate, and contract were quantified. An expression microarray, using 23,937 probes, and a pathway analysis were performed. Results: TM cells readily phagocytosed pigment granules. Pigment induced stress fiber formation (pigment (P): 60.1 ± 0.3%, n = 10, control (C): 38.4 ± 2.5%, n = 11, P < 0.001) and contraction at 24 hours onward (P < 0.01). Phagocytosis declined (P: 68.7 ± 1.3%, C: 37.0 ± 1.1%, n = 3, P < 0.001) and migration was reduced after 6 hours (P: 28.0.1 ± 2.3, n = 12, C: 40.6 ± 3.3, n = 13, P < 0.01). Microarray analysis revealed that Rho, IGF-1, and TGFβ signaling cascades were central to these responses. Conclusions: TM cell exposure to pigment dispersion resulted in reduced phagocytosis and migration, as well as increased stress fiber formation and cell contraction. The Rho signaling pathway played a central and early role, suggesting that its inhibitors could be used as a specific intervention in treatment of pigmentary glaucoma.

Additive manufacturing (3D printing) and computer-aided design (CAD) still have limited up-take in biomedical and bioengineering research and education, despite the significant potential of these technologies. The utility of organ-scale 3D-printed models of living structures is widely appreciated, while the workflows for microscopy data translation into tactile-accessible replicas are not well developed yet. Here, we demonstrate an accessible and reproducible CAD-based methodology for generating 3D-printed scalable models of human cells cultured in vitro and imaged using conventional scanning confocal microscopy and fused deposition modelling (FDM) 3D printing. We termed this technology CiTo-3DP (Cells-in-Touch for 3D Printing). As a proof-of-concept, we created CiTo-3DP models of human pancreatic cancer cells and healthy dermal fibroblasts by using selectively stained nuclei and the cytoskeleton components (f-actin and α-smooth muscle actin). The production of dismountable sets of cellular components was al-so shown. The CiTo-3DP approach can be adapted to comprehensively present various cell types, subcellular structures and extracellular matrices. We envisage that the resulting CAD and 3D printed models could be used for further applications, including but not limited to in silico simulations for biology, medicine, pharmacological research, tissue engineering, morphometrical analysis, multiphysics modelling, education, rehabilitation of visually impaired people, and integration into virtual reality.

Arf-like protein 2 (ARL2) is a ubiquitously expressed small GTPase with multiple functions. In cell culture, ARL2 participates with tubulin cofactor D (TBCD) in the neogenesis of tubulin αβ-heterodimers, the building blocks of microtubules. To evaluate this function in retina, we conditionally deleted ARL2 in mouse retina at two distinct stages, either during embryonic development (retArl2-/-) or after ciliogenesis specifically in rods (rodArl2-/-). retArl2-/- retina sections displayed distorted nuclear layers and a disrupted microtubule cytoskeleton (MTC) as early as postnatal day 6 (P6). Rod and cone outer segments (OS) did not form. By contrast, rod ARL2 knockouts were stable at postnatal day 35 and revealed normal ERG responses. Cytoplasmic dynein is reduced in retArl2-/- inner segments (IS), suggesting that dynein may be unstable in the absence of a normal MTC. We investigated microtubular stability in the absence of either ARL2 (retARL2-/-) or DYNC1H1 (retDync1h1-/-), the dynein heavy chain, and found that both the retArl2-/- and retDync1h1-/- retinas exhibited reduced microtubules and nuclear layer distortion. The results suggest that ARL2 and dynein depend on each other to generate a functional MTC during early photoreceptor development.

The cellular cytoskeleton provides the cell with a mechanical rigidity which allows mechanical interaction between cells and the extracellular environment. The actin structure plays a key role in mechanical events like motility, or establishment of cell polarity. From the earliest stages of development, as represented by ex vivo expansion of naïve embryonic stem cells (ESCs), the critical mechanical role of the actin structure is becoming recognized as a vital cue for correct segregation and lineage control of cells and as a regulatory structure that controls several transcription factors. Naïve ESCs have a characteristic morphology and the ultrastructure that underlies this condition remains to be further investigated. Here, we investigate the 3D actin cytoskeleton of naïve mouse ESCs using super resolution optical reconstruction microscopy (STORM). We investigate the morphological, cytoskeletal and mechanical changes in cells cultured in 2i or Serum/LIF media reflecting a homogenous preimplantation cell state and a state that is closer to embarking on differentiation. STORM imaging showed that the peripheral actin structure undergoes a dramatic change between the two media conditions. We also detected micro-rheological differences in the cell periphery between the cells cultured in these two media correlating well with the observed nano-architecture of the ESCs in the two different culture conditions. These results pave the way for linking physical properties and cytoskeletal architecture to cell morphology during early development.

At present more than 9 million people die of cancer every year. Simple and broad-spectrum drugs are still an urgent need for cancer patients. Recently, we proposed a new hypothesis that intracellular osmotic pressure (IOP) is the driving force of cell division, and abnormal tumor proliferation is the result of uncontrolled IOP in cells. On the one hand, aneuploidy and abnormal function of Na⁺/K⁺ pump lead to a faster rise of IOP in tumor cell than normal cells, on the other hand, abnormality of cytoskeleton assembly leads to the decrease of tolerance limit of cell membrane (TLCM) of tumor cells for resisting IOP. This hypothesis predicts: 1)Tumor cells were more intolerant to hypotonic stress than normal cells. 2) Maligancies may be sellectively killed by a suddenn increase of IOP and combined with decrease of the TLCM of tumors. Na⁺/K⁺ pump inhibitors can promotely increase the IOP of tumor cells and cytoskeleton inhibitors can dramatically lower the TLCM of tumor cells. Therefore, Na⁺/K⁺ pump and cytoskeleton inhibitors may have a synergetic effect to kill tumor cells. 3) Molecules regulating cell osmolality may be new targets for cancer treatment.

The cytoplasmic membrane is the fundamental component of all living cells, which participates in various physiological processes, such as material exchange, stress response, cell recognition, signal transduction, cellular immunity, apoptosis, pathogenicity, etc. The normal function of a cytoplasmic membrane requires stable organization of transmembrane protein-lipid microdomains, transmembrane protein-cell wall microdomains, and cytoskeleton-transmembrane protein microdomains. Here, we review the mechanisms and functions of various membrane lipid components, fatty acid content and saturation, membrane curvature, and cell wall and cytoskeleton in plasma membrane homeostasis affecting the pathogenicity of pathogenic fungi. Pathogenic fungi maintains plasma membrane homeostasis and contributes to fungal virulence by maintaining plasma membrane assembly, structural and functional integrity of pathogenic fungi at various stages of cell development through interactions among lipid components of cytoplasmic membranes, transmembrane proteins, cytoskeleton and cell wall components, etc.

Extracellular vesicle (EV) release, which occurs in most eukaryotic cells, has recently been associated with peptidylarginine deiminase (PAD)-driven protein deimination. Evidence points to the involvement of deiminated cytoskeletal proteins and changes in histone deimination. Both PADs and EVs are associated with various pathologies including cancers, autoimmune and neurodegenerative diseases. The elevated PAD expression observed in cancers may contribute to increase in EV shedding observed from cancer cells, contributing to cancer progression. Similarly, elevated PAD expression observed in neurodegenerative diseases may cause increased EV shedding and spread of neurodegenerative EV cargo, contributing to disease progression and pathologies. Pharmacological inhibition of PAD-mediated deimination using pan-PAD inhibitor Cl-amidine, reduced cellular EV release in prostate cancer cells, rendering them significantly more susceptible to chemotherapeutic drugs. Studies on models of central nervous system damage have demonstrated critical functional roles for PADs and neuroprotective effects using PAD inhibitors in vivo, while human neurodegenerative iPSC in vitro models showed evidence of increased protein deimination. Besides using refined PAD inhibitors to selectively manipulate EV biogenesis for novel combination therapies in cancer treatment, we also speculate how EV biogenesis could be targeted via the newly identified PAD-pathway to ameliorate neurodegenerative disease progression.

Navigating growth cones are exposed to multiple signals simultaneously and have to integrate competing cues into a coherent navigational response. Integration of guidance cues is traditionally thought to occur at the level of cytoskeletal dynamics. Drosophila studies indicate that cells exhibit a low level of continuous caspase protease activation, and that axon guidance cues can activate or suppress caspase activity. We base a model for axon guidance on these observations. By analogy with other systems in which caspase signaling has non-apoptotic functions, we propose that caspase signaling can either reinforce repulsion or negate attraction in response to external guidance cues by cleaving cytoskeletal proteins. Over the course of an entire trajectory, incorrectly navigating axons may pass the threshold for apoptosis and be eliminated, whereas axons making correct decisions will survive. These observations would also explain why neurotrophic factors can act as axon guidance cues and why axon guidance systems such as Slit/Robo signaling may act as tumor suppressors in cancer.