Quantitation of F-actin in cytoskeletal reorganization: Context, methodology and implications.

The actin cytoskeleton is involved in a large number of cellular signaling events in addition to providing structural integrity to the cell. Actin polymerization is a key event during cellular signaling. Although the role of actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, the reorganization of the actin cytoskeleton upon signaling has been rarely explored due to lack of suitable assays. Keeping in mind this lacuna, we developed a confocal microscopy based approach that relies on high magnification imaging of cellular F-actin, followed by image reconstruction using commercially available software. In this review, we discuss the context and relevance of actin quantitation, followed by a detailed hands-on approach of the methodology involved with specific points on troubleshooting and useful precautions. In the latter part of the review, we elucidate the method by discussing applications of actin quantitation from our work in several important problems in contemporary membrane biology ranging from pathogen entry into host cells, to GPCR signaling and membrane-cytoskeleton interaction. We envision that future discovery of cell-permeable novel fluorescent probes, in combination with genetically encoded actin-binding reporters, would allow real-time visualization of actin cytoskeleton dynamics to gain deeper insights into active cellular processes in health and disease.

Effects of cytochalasin D on relaxation process of skinned taenia cecum and carotid artery from guinea pig.

Actin linked regulatory mechanisms are known to contribute contraction/relaxation in smooth muscle. In order to clarify whether modulation of polymerization/depolymerization of actin filaments affects relaxation process, we examined the effects of cytochalasin D on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) taenia cecum and carotid artery preparations from guinea pigs. Cytochalasin D, an inhibitor of actin polymerization, significantly suppressed the force during relaxation both in skinned taenia cecum and carotid artery. The data fitting analysis of the relaxation processes indicates that cytochalasin D accelerates slow (latch-like) bridge dissociation. Cytochalasin D seems to directly disrupts actin filament organization or its length, resulting in modulation of actin filament structure that prevents myosin binding.

Oscillations of chlorophyll fluorescence after plasma membrane excitation in Chara originate from nonuniform composition of signaling metabolites in the streaming cytoplasm.

Excitable cells of higher plants and characean algae respond to stressful stimuli by generating action potentials (AP) whose regulatory influence on chlorophyll (Chl) fluorescence and photosynthesis extends over tens of minutes. Unlike plant leaves where the efficiency of photosystem II reaction (YII) undergoes a separate reversible depression after an individual AP, characean algae exhibit long-lasting oscillations of YII after firing AP, provided that Chl fluorescence is measured on microscopic cell regions. Internodal cells of charophytes feature an extremely fast cytoplasmic streaming that stops immediately during the spike and recovers within ~10 min after AP. In this study a possibility was examined that multiple oscillations of YII and Chl fluorescence parameters (F', Fm') result from the combined influence of metabolic rearrangements in chloroplasts and the cyclosis cessation-recovery cycle induced by the Ca2+ influx during AP. It is shown that the AP-induced Fm' and YII oscillations disappear when the fluidic communications between the analyzed area (AOI) and surrounding cell regions are restricted or eliminated. The microfluidic signaling was manipulated in two ways: by narrowing the illuminated cell area and by arresting the cytoplasmic streaming with cytochalasin D (CD). The inhibition of Fm' and YII oscillations was not caused by the loss of cell excitability, since CD-treated cells retained the capacity of AP generation. The mechanism of AP-induced oscillations of YII and Chl fluorescence seems to involve the lateral microfluidic transport of signaling substances in combination with the distribution pattern of these substances that was enhanced during the period of streaming cessation.

Enhancing suspended cell transfection by inducing localized distribution of the membrane actin cortex before exposure to electromechanical stimulation.

OBJECTIVES: During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection. RESULTS: A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation. CONCLUSIONS: Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.

Imaging Flow Cytometry of Multi-Nuclearity.

Multi-nuclearity is a common feature for cells in different cancers. Also, analysis of multi-nuclearity in cultured cells is widely used for evaluating the toxicity of different drugs. Multi-nuclear cells in cancer and under drug treatments form from aberrations in cell division and/or cytokinesis. These cells are a hallmark of cancer progression, and the abundance of multi-nucleated cells often correlates with poor prognosis.The use of standard bright field or fluorescent microscopy to analyze multi-nuclearity at the quantitative level is laborious and can suffer from user bias. Automated slide-scanning microscopy can eliminate scorer bias and improve data collection. However, this method has limitations, such as insufficient visibility of multiple nuclei in the cells attached to the substrate at low magnification.Since quantification of multi-nuclear cells using microscopic methods might be difficult, imaging flow cytometry (IFC) is a method of choice for this. We describe the experimental protocol for the preparation of the samples of multi-nucleated cells from the attached cultures and the algorithm for the analysis of these cells by IFC. Images of multi-nucleated cells obtained after mitotic arrest induced by taxol, as well as cells obtained after cytokinesis blockade by cytochalasin D treatment, can be acquired at a maximal resolution of IFC. We suggest two algorithms for the discrimination of single-nucleus and multi-nucleated cells. The advantages and disadvantages of IFC analysis of multi-nuclear cells in comparison with microscopy are discussed.

Wolbachia Promotes Its Own Uptake by Host Cells.

Wolbachia pipientis is an incredibly widespread bacterial symbiont of insects, present in an estimated 25 to 52% of species worldwide. Wolbachia is faithfully maternally transmitted both in a laboratory setting and in the wild. In an established infection, Wolbachia is primarily intracellular, residing within host-derived vacuoles that are associated with the endoplasmic reticulum. However, Wolbachia also frequently transfers between host species, requiring an extracellular stage to its life cycle. Indeed, Wolbachia has been moved between insect species for the precise goal of controlling populations. The use of Wolbachia in this application requires that we better understand how it initiates and establishes new infections. Here, we designed a novel method for live tracking Wolbachia cells during infection using a combination of stains and microscopy. We show that live Wolbachia cells are taken up by host cells at a much faster rate than dead Wolbachia cells, indicating that Wolbachia bacteria play a role in their own uptake and that Wolbachia colonization is not just a passive process. We also show that the host actin cytoskeleton must be intact for this to occur and that drugs that disrupt the actin cytoskeleton effectively abrogate Wolbachia uptake. The development of this live infection assay will assist in future efforts to characterize Wolbachia factors used during host infection.

Role of actin cytoskeleton in cargo delivery mediated by vertically aligned silicon nanotubes.

Nanofabrication technologies have been recently applied to the development of engineered nano-bio interfaces for manipulating complex cellular processes. In particular, vertically configurated nanostructures such as nanoneedles (NNs) have been adopted for a variety of biological applications such as mechanotransduction, biosensing, and intracellular delivery. Despite their success in delivering a diverse range of biomolecules into cells, the mechanisms for NN-mediated cargo transport remain to be elucidated. Recent studies have suggested that cytoskeletal elements are involved in generating a tight and functional cell-NN interface that can influence cargo delivery. In this study, by inhibiting actin dynamics using two drugs-cytochalasin D (Cyto D) and jasplakinolide (Jas), we demonstrate that the actin cytoskeleton plays an important role in mRNA delivery mediated by silicon nanotubes (SiNTs). Specifically, actin inhibition 12 h before SiNT-cellular interfacing (pre-interface treatment) significantly dampens mRNA delivery (with efficiencies dropping to 17.2% for Cyto D and 33.1% for Jas) into mouse fibroblast GPE86 cells, compared to that of untreated controls (86.9%). However, actin inhibition initiated 2 h after the establishment of GPE86 cell-SiNT interface (post-interface treatment), has negligible impact on mRNA transfection, maintaining > 80% efficiency for both Cyto D and Jas treatment groups. The results contribute to understanding potential mechanisms involved in NN-mediated intracellular delivery, providing insights into strategic design of cell-nano interfacing under temporal control for improved effectiveness.

Comparison of incorporation of wild type and mutated actins into sarcomeres in skeletal muscle cells: A fluorescence recovery after photobleaching study.

The α-actin mutation G15R in the nucleotide-binding pocket of skeletal muscle, causes severe actin myopathy in human skeletal muscles. Expressed in cultured embryonic quail skeletal myotubes, YFP-G15R-α-actin incorporates in sarcomeres in a pattern indistinguishable from wildtype YFP-α-actin. However, patches of YFP-G15R-α-actin form, resembling those in patients. Analyses with FRAP of incorporation of YFP-G15R-α-actin showed major differences between fast-exchanging plus ends of overlapping actin filaments in Z-bands, versus slow exchanging ends of overlapping thin filaments in the middle of sarcomeres. Wildtype skeletal muscle YFP-α-actin shows a faster rate of incorporation at plus ends of F-actin than at their minus ends. Incorporation of YFP-G15R-α-actin molecules is reduced at plus ends, increased at minus ends. The same relationship of wildtype YFP-α-actin incorporation is seen in myofibrils treated with cytochalasin-D: decreased dynamics at plus ends, increased dynamics at minus ends, and F-actin aggregates. Speculation: imbalance of normal polarized assembly of F-actin creates excess monomers that form F-actin aggregates. Two other severe skeletal muscle YFP-α-actin mutations (H40Y and V163L) not in the nucleotide pocket do not affect actin dynamics, and lack F-actin aggregates. These results indicate that normal α-actin plus and minus end dynamics are needed to maintain actin filament stability, and avoid F-actin patches.

Elevated Pre- and Postoperative ROTEM™ Clot Lysis Indices Indicate Reduced Clot Retraction and Increased Mortality in Patients Undergoing Liver Transplantation.

Background: The ROTEM™ clot lysis index, describing the decrease in firmness of a clot with time, predicts mortality in various settings. The variability of the clot lysis index in surgical procedures and the involved pathophysiological mechanisms are unknown. We therefore compared pre- and postoperative clot lysis indices in liver transplantation (LTX) procedures, determined the eventual association with mortality, and investigated the mechanisms underlying decreased clot lysis index using inhibitors of fibrinolysis and clot retraction, respectively. Methods: In this retrospective cohort study, data on pre- and post-transplant ROTEM™ findings as obtained with EXTEM (tissue factor activation), INTEM (intrinsic system activation), FIBTEM (extrinsic system activation and inhibition of clot retraction), APTEM (extrinsic system activation and fibrinolysis inhibition), conventional laboratory coagulation tests, blood loss, transfusion of blood products, and outcome were registered. Results: Pre-transplant clot lysis indices showed a broad distribution ranging from 75% to 99% independent of the activator used (EXTEM, INTEM). During the surgical procedure, median clot lysis index values markedly increased from 92% to 97% (EXTEM) and 93% to 98% (INTEM), respectively (p < 0.0001 each). Aprotinin had no effect on either pre- or postsurgical clot lysis indices. Inhibition of platelet clot retraction with cytochalasin D (FIBTEM) markedly increased the preoperative clot lysis index. High pre- and post-transplantation clot lysis indices were associated with increased mortality irrespective of the activator used (EXTEM, INTEM) and the inhibition of fibrinolysis (APTEM). Inhibition of clot retraction (FIBTEM) abolished the association of clot lysis index with mortality in both pre- and post-transplantation samples. Conclusion: Both pre- and postoperative ROTEM™ clot lysis indices predict mortality in patients following liver transplantation. Inhibitor experiments reveal that the clot lysis index is not an indicator of fibrinolysis, but indicates platelet clot retraction. The marked increase of clot lysis index during liver transplantation is caused by a decrease in clot retraction with eventual consequences for clot stability, retraction of wound margins, and reperfusion of vessels in case of thrombosis.

Assessing Als3 Peptide-Binding Cavity and Amyloid-Forming Region Contributions to Candida albicans Invasion of Human Oropharyngeal Epithelial Cells.

Although it is widely recognized that disruption of ALS3 reduces the invasion of Candida albicans germ tubes into mammalian oral epithelial cells, the mechanism of this interaction was unexplored. C. albicans strains with structurally informed mutations to remove adhesive activity of the peptide-binding cavity (PBC) or aggregative activity mediated by the amyloid-forming region (AFR) were assessed for their ability to invade cultured human oropharyngeal epithelial cells. Initial assays utilized untreated fungal and epithelial cells. Subsequent work used epithelial cells treated with cytochalasin D and C. albicans cells treated with thimerosal to investigate invasion mediated by active penetration of germ tubes and epithelial cell induced endocytosis, respectively. Results demonstrated the importance of the PBC for the invasion process: loss of PBC function resulted in the same reduced-invasion phenotype as a C. albicans strain that did not produce Als3 on its surface. Invasion via active penetration was particularly compromised without PBC function. Loss of AFR function produced a wild-type phenotype in the untreated and thimerosal-treated invasion assays but increased invasion in cytochalasin D-treated epithelial cells. In previous work, reduced AFR-mediated Als3 aggregation increased C. albicans adhesion to cultured epithelial cell monolayers, presumably via increased PBC accessibility for ligand binding. Collectively, results presented here demonstrate that Als3 PBC-mediated adhesion is integral to its invasive function. These new data add to the mechanistic understanding of the role of Als3 in C. albicans invasion into mammalian oral epithelial cells.

Engineering a 3D In Vitro Model of Human Gingival Tissue Equivalent with Genipin/Cytochalasin D.

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air-liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.

Artificial Activation of Murine Oocytes Using Strontium to Derive Haploid and Diploid Parthenotes.

Parthenogenesis is a common reproductive strategy among lower animals that involves the development of an embryo from an oocyte, without any contribution from spermatozoon. This phenomenon does not occur naturally in placental mammals. However, the mammalian oocytes can be artificially activated in vitro using mechanical, electrical, and chemical stimuli which can develop up to the blastocyst stage. In this chapter, we describe the protocol for generating haploid and diploid parthenotes from mouse oocytes using strontium as the activating agent under in vitro conditions.

Ultrastructure of Myzocytosis and Cyst Formation, and the Role of Actin in Tubular Tether Formation in Colpodella sp. (ATCC 50594).

Free-living relatives of the Apicomplexa such as Colpodella species, Alphamonas species, and Voromonas pontica are predators that prey on ciliate, bodonid, and algal prey using the process of myzocytosis. During myzocytosis, the pseudoconoid is used to attach to the prey leading to aspiration of cytoplasmic contents of the prey into a posterior food vacuole formed in the predator, aided by secretions from the apical complex organelles. The conoid and associated proteins are conserved among the apicomplexa. However, the organization and function of the pseudoconoid during myzocytosis are not well understood. In this study, we investigated the morphology and ultrastructure of Colpodella sp. (ATCC 50594) during the stages of myzocytosis and cyst formation in the life cycle using light microscopy and transmission electron microscopy (TEM) in order to identify the organization of the tubular tether involved in nutrient aspiration by Colpodella sp. Tubular tethers of varying lengths were identified by light microscopy. We report that initial contact by Colpodella sp. trophozoites with Parabodo caudatus prey is by an area posterior to the apical tip of the rostrum that engulfs the membrane of the prey pulling it into the cytoplasm of the predator. The tubular tether that forms contains membranes of both predator and prey and is facilitated by microtubule organization and the cytoskeleton at the point of contact. Cytochalasin D treatment of diprotist cultures resulted in morphological distortions of trophozoites and the tubular tether suggesting a role of actin in the formation of the tubular tether. This mechanism of predation may provide insight into the mode of invasion observed in pathogenic apicomplexan zoites during host cell entry.

Actin Modulation Regulates the Alpha-1-Syntrophin/p66Shc Mediated Redox Signaling Contributing to the RhoA GTPase Protein Activation in Breast Cancer Cells.

SNTA1 signaling axis plays an essential role in cytoskeletal organization and is also implicated in breast cancers. In this study, we aimed to investigate the involvement of actin cytoskeleton in the propagation of SNTA1/p66shc mediated pro-metastatic cascade in breast cancer cells.The effect of actin filament depolymerization on SNTA1-p66Shc interaction and the trimeric complex formation was analyzed using co-immunoprecipitation assays. Immunofluorescence and RhoA activation assays were used to show the involvement of SNTA1-p66Shc interaction in RhoA activation and F-actin organization. Cellular proliferation and ROS levels were assessed using MTT assay and Amplex red catalase assay. The migratory potential was evaluated using transwell migration assay and wound healing assay.We found that cytochalasin D mediated actin depolymerization significantly declines endogenous interaction between SNTA1 and p66Shc protein in MDA-MB-231 cells. Results indicate that SNTA1 and p66Shc interact with RhoA protein under physiological conditions. The ROS generation and RhoA activation were substantially enhanced in cells overexpressing SNTA1 and p66Shc, promoting proliferation and migration in these cells. In addition, we found that loss of SNTA1-p66Shc interaction impaired actin organization, proliferation, and migration in breast cancer cells. Our results demonstrate a novel reciprocal regulatory mechanism between actin modulation and SNTA1/p66Shc/RhoA signaling cascade in human metastatic breast cancer cells.

Cyclic mechanical strain with high-tensile triggers autophagy in growth plate chondrocytes.

BACKGROUND: Mechanical loading has been widely considered to be essential for growth plate to maintain metabolism and development. Cyclic mechanical strain has been demonstrated to induce autophagy, whereas the relationship between cyclic tensile strain (CTS) and autophagy in growth plate chondrocytes (GPCs) is not clear. The objective of this study was to investigate whether CTS can regulate autophagy in GPCs in vitro and explore the potential mechanisms of this regulation. METHODS: The 2-week-old Sprague-Dawley rat GPCs were subjected to CTS of varying magnitude and duration at a frequency of 2.0 Hz. The mRNA levels of autophagy-related genes were measured by RT-qPCR. The autophagy in GPCs was verified by transmission electron microscopy (TME), immunofluorescence and Western blotting. The fluorescence-activated cell sorting (FACS) was employed to detect the percentage of apoptotic and necrotic cells. RESULTS: In GPCs, CTS significantly increased the mRNA and protein levels of autophagy-related genes, such as LC3, ULK1, ATG5 and BECN1 in a magnitude- and time-dependent manner. There was no significant difference in the proportion of apoptotic and necrotic cells between control group and CTS group. The autophagy inhibitors, 3-methyladenine (3MA) and chloroquine (CQ) reversed the CTS-induced autophagy via promoting the formation of autophagosomes. Cytochalasin D (cytoD), an inhibitor of G-actin polymerization into F-actin, could effectively block the CTS-induced autophagy in GPCs. CONCLUSION: Cyclic mechanical strain with high-tensile triggers autophagy in GPCs, which can be suppressed by 3MA and CQ, and cytoskeletal F-actin microfilaments organization plays a key role in chondrocytes' response to mechanical loading.

Integrity of the Actin Cytoskeleton of Host Macrophages is Necessary for Mycobacterial Entry.

Macrophages are the primary hosts for Mycobacterium tuberculosis (M. tb), an intracellular pathogen, and the causative organism of tuberculosis (TB) in humans. While M. tb has the ability to enter and survive in host macrophages, the precise mechanism of its internalization, and factors that control this essential process are poorly defined. We have previously demonstrated that perturbations in levels of cholesterol and sphingolipids in macrophages lead to significant reduction in the entry of Mycobacterium smegmatis (M. smegmatis), a surrogate model for mycobacterial internalization, signifying a role for these plasma membrane lipids in interactions at the host-pathogen interface. In this work, we investigated the role of the host actin cytoskeleton, a critical protein framework underlying the plasma membrane, in the entry of M. smegmatis into human macrophages. Our results show that cytochalasin D mediated destabilization of the actin cytoskeleton of host macrophages results in a dose-dependent reduction in the entry of mycobacteria. Notably, the internalization of Escherichia coli remained invariant upon actin destabilization of host cells, implying a specific involvement of the actin cytoskeleton in mycobacterial infection. By monitoring the F-actin content of macrophages utilizing a quantitative confocal microscopy-based technique, we observed a close correlation between the entry of mycobacteria into host macrophages with cellular F-actin content. Our results constitute the first quantitative analysis of the role of the actin cytoskeleton of human macrophages in the entry of mycobacteria, and highlight actin-mediated mycobacterial entry as a potential target for future anti-TB therapeutics.

The Role of Cytoskeletal Proteins in the Formation of a Functional In Vitro Blood-Brain Barrier Model.

The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain's microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.

Actin and Microtubules Differently Contribute to Vacuolar Targeting Specificity during the Export from the ER.

Plants rely on both actin and microtubule cytoskeletons to fine-tune sorting and spatial targeting of membranes during cell growth and stress adaptation. Considerable advances have been made in recent years in the comprehension of the relationship between the trans-Golgi network/early endosome (TGN/EE) and cytoskeletons, but studies have mainly focused on the transport to and from the plasma membrane. We address here the relationship of the cytoskeleton with different endoplasmic reticulum (ER) export mechanisms toward vacuoles. These emergent features of the plant endomembrane traffic are explored with an in vivo approach, providing clues on the traffic regulation at different levels beyond known proteins' functions and interactions. We show how traffic of vacuolar markers, characterized by different vacuolar sorting determinants, diverges at the export from the ER, clearly involving different components of the cytoskeleton.

Early endocytosis as a key to understanding mechanisms of plasma membrane tension regulation in filamentous fungi.

BACKGROUND INFORMATION: Two main systems regulate plasma membrane tension (PMT) and provide a close connection between the protoplast and the cell wall in fungi: turgor pressure and the actin cytoskeleton. These systems work together with the plasma membrane focal adhesion to the cell wall and their contribution to fungal cell organization and physiology has been partially studied. However, it remains controversial in model filamentous ascomycetes and oomycetes and even less investigated in filamentous basidiomycetes. Early endocytosis can be used to research the mechanisms regulating PMT since the dynamics of early endocytosis is largely dependent on this tension. RESULTS: This study examined the effects of actin polymerization inhibitors and hyperosmotic shock on early endocytosis and cell morphology in two filamentous basidiomycetes. The main obtained results are: (i) the depolymerisation of F-actin leads to the fast formation of endocytic pits while inhibiting of their scission from the plasma membrane and (ii) the moderate hyperosmotic shock does not affect the dynamics of early endocytosis. These and our other results have allowed suggesting a curtain model for the regulation of PMT in basidiomycetes. CONCLUSIONS AND SIGNIFICANCE: According to the proposed curtain model, the PMT in many non-apical cells of hyphae is more often regulated not by turgor pressure but by a system of actin driver cables that are associated with the proteins of the focal adhesion sites. The change in PMT occurs similar to the movement of a curtain along the curtain rod using the curtain drivers. This model addresses the fundamental properties of the fungal structure and physiology. It requires confirmation including the currently technically unavailable high-quality labelling of the actin cytoskeleton of the basidiomycetes.

Actomyosin-dependent invasion of endothelial sprouts in collagen.

During sprouting angiogenesis-the growth of blood vessels from the existing vasculature-endothelial cells (ECs) adopt an elongated invasive form and exert forces at cell-cell and cell-matrix interaction sites. These cell shape changes and cellular tractions require extensive reorganizations of the actomyosin network. However, the respective roles of actin and myosin for endothelial sprouting are not fully elucidated. In this study, we further investigate these roles by treating 2D-migrating and 3D-sprouting ECs with chemical compounds targeting either myosin or actin. These treatments affected the endothelial cytoskeleton drastically and reduced the invasive response in a compound-specific manner; pointing toward a tight control of the actin and myosin activity during sprouting. Clusters in the data further illustrate that endothelial sprout morphology is sensitive to the in vitro model mechanical microenvironment and directs future research toward mechanical substrate guidance as a strategy for promoting engineered tissue vascularization. In summary, our results add to a growing corpus of research highlighting a key role of the cytoskeleton for sprouting angiogenesis.