Actin-associated Proteins Research Articles

Cell-to-Cell Connectivity Assays for the Analysis of Cytoskeletal and Other Regulators of Plasmodesmata.

The actin cytoskeleton has close but so far incompletely understood connections to plasmodesmata, the cell junctions of plants. Plasmodesmata are essential for plant development and responses to biotic and abiotic stresses and facilitate the intercellular exchange of metabolites and hormones but also macromolecules such as proteins and RNAs. The molecular size exclusion limited of plasmodesmata is dynamically regulated, including by actin-associated proteins. Therefore, experimental analysis of plasmodesmal regulation can be relevant to plant cytoskeleton research. This chapter presents two simple imaging-based protocols for analyzing macromolecular cell-to-cell connectivity in leaves.

Minimal Kidney Disease Phenotype in Shroom3 Heterozygous Null Mice.

Shroom family member 3 (SHROOM3) encodes an actin-associated protein that regulates epithelial morphology during development. Several genome-wide association studies (GWAS) have identified genetic variances primarily in the 5' region of SHROOM3, associated with chronic kidney disease (CKD) and poor transplant outcomes. These genetic variants are associated with alterations in Shroom3 expression. Characterize the phenotypic abnormalities associated with reduced Shroom3 expression in postnatal day 3-, 1-month and 3-month-old mice. The Shroom3 protein expression pattern was determined by immunofluorescence. We generated Shroom3 heterozygous null mice (Shroom3Gt/+) and performed comparative analyses with wild type littermates based on somatic and kidney growth, gross renal anatomy, renal histology, renal function at postnatal day 3, 1 month, and 3 months. The Shroom3 protein expression localized to the apical regions of medullary and cortical tubular epithelium in postnatal wild type kidneys. Co-immunofluorescence studies confirmed protein expression localized to the apical side of the tubular epithelium in proximal convoluted tubules, distal convoluted tubules, and collecting ducts. While Shroom3 heterozygous null mice exhibited reduced Shroom3 protein expression, no differences in somatic and kidney growth were observed when compared to wild type mice. Although, rare cases of unilateral hypoplasia of the right kidney were observed at postnatal 1 month in Shroom3 heterozygotes. Yet renal histological analysis did not reveal any overt abnormalities in overall kidney structure or in glomerular and tubular organization in Shroom3 heterozygous null mice when compared to wild type mice. Analysis of the apical-basolateral orientation of the tubule epithelium demonstrated alterations in the proximal convoluted tubules and modest disorganization in the distal convoluted tubules at 3 months in Shroom3 heterozygotes. Additionally, these modest abnormalities were not accompanied by tubular injury or physiological defects in renal and cardiovascular function. Taken together, our results describe a mild kidney disease phenotype in adult Shroom3 heterozygous null mice, suggesting that Shroom3 expression and function may be required for proper structure and maintenance of the various tubular epithelial parenchyma of the kidney.

Quantitative Investigation of the Link between Actin Cytoskeleton Dynamics and Cellular Behavior.

Actin cytoskeleton reorganization, which is governed by actin-associated proteins, has a close relationship with the change of cell biological behavior. However, a perceived understanding of how actin mechanical property links to cell biological property remains unclear. This paper reports a label-free biomarker to indicate this interrelationship by using the actin cytoskeleton model and optical tweezers (OT) manipulation technology. Both biophysical and biochemical methods were employed, respectively, as stimuli for two case studies. By comparing the mechanical and biological experiment results of the leukemia cells under electrical field exposure and human mesenchymal stem cells (hMSC) under adipogenesis differentiation, we concluded that β-actin can function as an indicator in characterizing the alteration of cellular biological behavior during the change of actin cytoskeleton mechanical property. This study demonstrated an effective way to probe a quantitative understanding of how actin cytoskeleton reorganization reflects the interrelation between cell mechanical property and cell biological behavior.

TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway

BackgroundMicroglia, the innate immune cells in the central nervous system, play an essential role in brain homeostasis, neuroinflammation and brain infections. Dysregulated microglia, on the other hand, are associated with neurodegenerative diseases, yet the mechanisms underlying pro-inflammatory gene expression in microglia are incompletely understood.MethodsWe investigated the role of the actin-associated protein tropomyosin 1 (TPM1) in regulating pro-inflammatory phenotype of microglia in the retina by using a combination of cell culture, immunocytochemistry, Western blot, qPCR, TUNEL, RNA sequencing and electroretinogram analysis. TREM2−/− mice were used to investigate whether TPM1 regulated pro-inflammatory responses downstream of TREM2. To conditionally deplete microglia, we backcrossed CX3CR1CreER mice with Rosa26iDTR mice to generate CX3CR1CreER:Rosa26iDTR mice.ResultsWe revealed a vital role for TPM1 in regulating pro-inflammatory phenotype of microglia. We found that TPM1 drove LPS-induced inflammation and neuronal death in the retina via the PKA/CREB pathway. TPM1 knockdown ameliorated LPS-induced inflammation in WT retinas yet exaggerated the inflammation in TREM2−/− retinas. RNA sequencing revealed that genes associated with M1 microglia and A1 astrocytes were significantly downregulated in LPS-treated WT retinas but upregulated in LPS-treated TREM2−/− retinas after TPM1 knockdown. Mechanistically, we demonstrated that CREB activated by TPM1 knockdown mediated anti-inflammatory genes in LPS-treated WT retinas but pro-inflammatory genes in LPS-treated TREM2−/− retinas, suggesting a novel role for TREM2 as a brake on TPM1-mediated inflammation. Furthermore, we identified that TPM1 regulated inflammation downstream of TREM2 and in a microglia-dependent manner.ConclusionsWe demonstrate that TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway. Our findings suggest that TPM1 could be a potential target for therapeutic intervention in brain diseases.

PFN1 Inhibits Myogenesis of Bovine Myoblast Cells via Cdc42-PAK/JNK.

Myoblast differentiation is essential for the formation of skeletal muscle myofibers. Profilin1 (Pfn1) has been identified as an actin-associated protein, and has been shown to be critically important to cellular function. Our previous study found that PFN1 may inhibit the differentiation of bovine skeletal muscle satellite cells, but the underlying mechanism is not known. Here, we confirmed that PFN1 negatively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Immunoprecipitation assay combined with mass spectrometry showed that Cdc42 was a binding protein of PFN1. Cdc42 could be activated by PFN1 and could inhibit the myogenic differentiation like PFN1. Mechanistically, activated Cdc42 increased the phosphorylation level of p2l-activated kinase (PAK), which further activated the phosphorylation activity of c-Jun N-terminal kinase (JNK), whereas PAK and JNK are inhibitors of myogenic differentiation. Taken together, our results reveal that PFN1 is a repressor of bovine myogenic differentiation, and provide the regulatory mechanism.

Cytoskeletal dysregulation and neurodegenerative disease: Formation, monitoring, and inhibition of cofilin-actin rods.

The presence of atypical cytoskeletal dynamics, structures, and associated morphologies is a common theme uniting numerous diseases and developmental disorders. In particular, cytoskeletal dysregulation is a common cellular feature of Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. While the numerous activators and inhibitors of dysregulation present complexities for characterizing these elements as byproducts or initiators of the disease state, it is increasingly clear that a better understanding of these anomalies is critical for advancing the state of knowledge and plan of therapeutic attack. In this review, we focus on the hallmarks of cytoskeletal dysregulation that are associated with cofilin-linked actin regulation, with a particular emphasis on the formation, monitoring, and inhibition of cofilin-actin rods. We also review actin-associated proteins other than cofilin with links to cytoskeleton-associated neurodegenerative processes, recognizing that cofilin-actin rods comprise one strand of a vast web of interactions that occur as a result of cytoskeletal dysregulation. Our aim is to present a current perspective on cytoskeletal dysregulation, connecting recent developments in our understanding with emerging strategies for biosensing and biomimicry that will help shape future directions of the field.

Direct observation of the molecular mechanism underlying protein polymerization.

Protein assembly is a main route to generating complexity in living systems. Revealing the relevant molecular details is challenging because of the intrinsic heterogeneity of species ranging from few to hundreds of molecules. Here, we use mass photometry to quantify and monitor the full range of actin oligomers during polymerization with single-molecule sensitivity. We find that traditional nucleation-based models cannot account for the observed distributions of actin oligomers. Instead, the key step of filament formation is a slow transition between distinct states of an actin filament mediated by cation exchange or ATP hydrolysis. The resulting model reproduces important aspects of actin polymerization, such as the critical concentration for filament formation and bulk growth behavior. Our results revise the mechanism of actin nucleation, shed light on the role and function of actin-associated proteins, and introduce a general and quantitative means to studying protein assembly at the molecular level.

EPLINβ Is Involved in the Assembly of Cadherin-catenin Complexes in Osteoblasts and Affects Bone Formation.

Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINβ. In this study, we investigated the role of EPLINβ in osteoblasts using EPLINβ-deficient (EPLINβGT/GT) mice. The skeletal phenotype of EPLINβGT/GT mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in EPLINβGT/GT mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in EPLINβGT/GT mice, suggesting aberrant cell adhesion. In EPLINβGT/GT osteoblasts, α- and β-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINβ knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as RUNX2, Osterix, ALP, and Col1a1 mRNA were reduced in EPLINβ knockdown cells, suggesting an important role for EPLINβ in osteoblast formation. In conclusion, we propose that EPLINβ is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.

The Actin Cytoskeleton Responds to Inflammatory Cues and Alters Macrophage Activation.

Much remains to be learned about the molecular mechanisms underlying a class of human disorders called actinopathies. These genetic disorders are characterized by loss-of-function mutations in actin-associated proteins that affect immune cells, leading to human immunopathology. However, much remains to be learned about how cytoskeletal dysregulation promotes immunological dysfunction. The current study reveals that the macrophage actin cytoskeleton responds to LPS/IFNγ stimulation in a biphasic manner that involves cellular contraction followed by cellular spreading. Myosin II inhibition by blebbistatin blocks the initial contraction phase and lowers iNOS protein levels and nitric oxide secretion. Conversely, conditional deletion of Arp2/3 complex in macrophages attenuates spreading and increases nitric oxide secretion. However, iNOS transcription is not altered by loss of myosin II or Arp2/3 function, suggesting post-transcriptional regulation of iNOS by the cytoskeleton. Consistent with this idea, proteasome inhibition reverses the effects of blebbistatin and rescues iNOS protein levels. Arp2/3-deficient macrophages demonstrate two additional phenotypes: defective MHCII surface localization, and depressed secretion of the T cell chemokine CCL22. These data suggest that interplay between myosin II and Arp2/3 influences macrophage activity, and potentially impacts adaptive-innate immune coordination. Disrupting this balance could have detrimental impacts, particularly in the context of Arp2/3-associated actinopathies.

Localization of host endocytic and actin-associated proteins during Shigella flexneri intracellular motility and intercellular spreading.

Shigella flexneri (S. flexneri), the causative agent of bacillary dysentery, uses an effector-mediated strategy to hijack host cells and cause disease. To propagate and spread within human tissues, S. flexneri bacteria commandeer the host actin cytoskeleton to generate slender actin-rich comet tails to move intracellularly, and later, plasma membrane actin-based protrusions to move directly between adjacent host cells. To facilitate intercellular bacterial spreading, large micron-sized endocytic-like membrane invaginations form at the periphery of neighboring host cells that come into contact with S. flexneri-containing membrane protrusions. While S. flexneri comet tails and membrane protrusions consist primarily of host actin cytoskeletal proteins, S. flexneri membrane invaginations remain poorly understood with only clathrin and the clathrin adapter epsin-1 localized to the structures. Tangentially, we recently reported that Listeria monocytogenes, another actin-hijacking pathogen, exploits an assortment of caveolar and actin-bundling proteins at their micron-sized membrane invaginations formed during their cell-to-cell movement. Thus, to further characterize the S. flexneri disease process, we set out to catalog the distribution of a variety of actin-associated and caveolar proteins during S. flexneri actin-based motility and cell-to-cell spreading. Here we show that actin-associated proteins found at L. monocytogenes comet tails and membrane protrusions mimic those present at S. flexneri comet tails with the exception of α-actinins 1 and 4, which were shed from S. flexneri membrane protrusions. We also demonstrate that all known host endocytic components found at L. monocytogenes membrane invaginations are also present at those formed during S. flexneri infections.

Proteomic Profiling and Pathway Analysis of Acid Stress-Induced Vasorelaxation of Mesenteric Arteries In Vitro.

Although metabolic acidosis is associated with numerous pathophysiological conditions and its vasorelaxation effects have been well described in different animal and culture models, the molecular mechanisms of acidosis-induced vasorelaxation are not fully understood. Mesenteric artery models have been used extensively to examine the vascular response to various pathophysiological conditions. Our previous studies and several other reports have suggested the vascular responses of goat mesenteric arteries and human arteries to various stimuli, including acidic stress, are highly similar. In this study, to further identify the signaling molecules responsible for altered vasoreactivity in response to acidic pH, we examined the proteomic profile of acid stress-induced vasorelaxation using a goat mesenteric artery model. The vascular proteomes under acidic pH were compared using 2D-GE with 7 cm IPG strips and mini gels, LC-MS/MS, and MALDI TOF MS. The unique proteins identified by mass spectroscopy were actin, transgelin, WD repeat-containing protein 1, desmin, tropomyosin, ATP synthase β, Hsp27, aldehyde dehydrogenase, pyruvate kinase, and vitamin K epoxide reductase complex subunit 1-like protein. Out of five protein spots identified as actin, three were upregulated > 2-fold. ATP synthase β was also upregulated (2.14-fold) under acid stress. Other actin-associated proteins upregulated were transgelin, desmin, and WD repeat-containing protein 1. Isometric contraction studies revealed that both receptor-mediated (histamine) and non-receptor-mediated (KCl) vasocontraction were attenuated, whereas acetylcholine-induced vasorelaxation was augmented under acidosis. Overall, the altered vasoreactivity under acidosis observed in the functional studies could possibly be attributed to the increase in expression of actin and ATP synthase β.

Myosin 1D and the branched actin network control the condensation of p62 bodies.

Biomolecular condensation driven by liquid-liquid phase separation (LLPS) is key to assembly of membraneless organelles in numerous crucial pathways. It is largely unknown how cellular structures or components spatiotemporally regulate LLPS and condensate formation. Here we reveal that cytoskeletal dynamics can control the condensation of p62 bodies comprising the autophagic adaptor p62/SQSTM1 and poly-ubiquitinated cargos. Branched actin networks are associated with p62 bodies and are required for their condensation. Myosin 1D, a branched actin-associated motor protein, drives coalescence of small nanoscale p62 bodies into large micron-scale condensates along the branched actin network. Impairment of actin cytoskeletal networks compromises the condensation of p62 bodies and retards substrate degradation by autophagy in both cellular models and Myosin 1D knockout mice. Coupling of LLPS scaffold to cytoskeleton systems may represent a general mechanism by which cells exert spatiotemporal control over phase condensation processes.

Disc and Actin Associated Protein 1 influences attachment in the intestinal parasite Giardia lamblia.

The deep-branching eukaryote Giardia lamblia is an extracellular parasite that attaches to the host intestine via a microtubule-based structure called the ventral disc. Control of attachment is mediated in part by the movement of two regions of the ventral disc that either permit or exclude the passage of fluid under the disc. Several known disc-associated proteins (DAPs) contribute to disc structure and function, but no force-generating protein has been identified among them. We recently identified several Giardia actin (GlActin) interacting proteins at the ventral disc, which could potentially employ actin polymerization for force generation and disc conformational changes. One of these proteins, Disc and Actin Associated Protein 1 (DAAP1), is highly enriched at the two regions of the disc previously shown to be important for fluid flow during attachment. In this study, we investigate the role of both GlActin and DAAP1 in ventral disc morphology and function. We confirmed interaction between GlActin and DAAP1 through coimmunoprecipitation, and used immunofluorescence to localize both proteins throughout the cell cycle and during trophozoite attachment. Similar to other DAPs, the association of DAAP1 with the disc is stable, except during cell division when the disc disassembles. Depletion of GlActin by translation-blocking antisense morpholinos resulted in both impaired attachment and defects in the ventral disc, indicating that GlActin contributes to disc-mediated attachment. Depletion of DAAP1 through CRISPR interference resulted in intact discs but impaired attachment, gating, and flow under the disc. As attachment is essential for infection, elucidation of these and other molecular mediators is a promising area for development of new therapeutics against a ubiquitous parasite.

Neflamapimod induces vasodilation in resistance mesenteric arteries by inhibiting p38 MAPK\u03b1 and downstream Hsp27 phosphorylation

Neflamapimod, a selective inhibitor of p38 mitogen activated protein kinase alpha (MAPKα), is under clinical investigation for its efficacy in Alzheimer’s disease (AD) and dementia with Lewy Bodies (DLB). Here, we investigated if neflamapimod-mediated acute inhibition of p38 MAPKα could induce vasodilation in resistance-size rat mesenteric arteries. Our pressure myography data demonstrated that neflamapimod produced a dose-dependent vasodilation in mesenteric arteries. Our Western blotting data revealed that acute neflamapimod treatment significantly reduced the phosphorylation of p38 MAPKα and its downstream target heat-shock protein 27 (Hsp27) involved in cytoskeletal reorganization and smooth muscle contraction. Likewise, non-selective inhibition of p38 MAPK by SB203580 attenuated p38 MAPKα and Hsp27 phosphorylation, and induced vasodilation. Endothelium denudation or pharmacological inhibition of endothelium-derived vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) had no effect on such vasodilation. Neflamapimod-evoked vasorelaxation remained unaltered by the inhibition of smooth muscle cell K+ channels. Altogether, our data for the first time demonstrates that in resistance mesenteric arteries, neflamapimod inhibits p38 MAPKα and phosphorylation of its downstream actin-associated protein Hsp27, leading to vasodilation. This novel finding may be clinically significant and is likely to improve systemic blood pressure and cognitive deficits in AD and DLB patients for which neflamapimod is being investigated.

Effect of Ezrin on regulating trophoblast cell invasion via PKC signaling pathway in unexplained recurrent spontaneous abortion

Trophoblast cells are the most important cells in early pregnancy and their invasion are essential to the establishment and maintenance of pregnancy. Inadequate trophoblast cell invasion has been closely associated with several pregnancy-associated diseases including recurrent spontaneous abortion (RSA). Ezrin is an actin-associated protein, known as a marker for carcinogenesis and metastasis in solid tumors, has been proposed to play a role in the formation of microvilli in the early embryo. To further characterize its function in early pregnancy, we explored the expression of Ezrin in the trophoblast cells in early pregnancy. In this study, compared with normal pregnant women, we demonstrated that the expression of Ezrin and phosphorylated Ezrin decreased in the trophoblast cells in unexplained RSA (URSA) patients, and knockdown of Ezrin expression could suppress the invasiveness of trophoblast cells significantly. Various studies indicated that the phosphorylation of Ezrin on C-terminal threonine residue (T567) is a key event in the regulation of its activity. Our further exploration indicated that Ezrin was activated via PKC pathway. Furthermore, inhibition of the PKC pathway by a specific inhibitor suppressed invasiveness of Bewo cells. On the other hand, activation of the PKC pathway could increase the relative capacity of trophoblast cell invasion, while Ezrin knockdown reversed PKC activation induced cell invasion. These findings might provide a new fundamental mechanism for successful pregnancy and new diagnostic and therapeutic target for RSA.

Actin-Associated Proteins and Small Molecules Targeting the Actin Cytoskeleton.

Actin-associated proteins (AAPs) act on monomeric globular actin (G-actin) and polymerized filamentous actin (F-actin) to regulate their dynamics and architectures which ultimately control cell movement, shape change, division; organelle localization and trafficking. Actin-binding proteins (ABPs) are a subset of AAPs. Since actin was discovered as a myosin-activating protein (hence named actin) in 1942, the protein has also been found to be expressed in non-muscle cells, and numerous AAPs continue to be discovered. This review article lists all of the AAPs discovered so far while also allowing readers to sort the list based on the names, sizes, functions, related human diseases, and the dates of discovery. The list also contains links to the UniProt and Protein Atlas databases for accessing further, related details such as protein structures, associated proteins, subcellular localization, the expression levels in cells and tissues, mutations, and pathology. Because the actin cytoskeleton is involved in many pathological processes such as tumorigenesis, invasion, and developmental diseases, small molecules that target actin and AAPs which hold potential to treat these diseases are also listed.

Accumulation of systematic TPM1 mediates inflammation and neuronal remodeling by phosphorylating PKA and regulating the FABP5/NF-κB signaling pathway in the retina of aged mice.

The molecular mechanisms underlying functional decline during normal brain aging are poorly understood. Here, we identified the actin‐associated protein tropomyosin 1 (TPM1) as a new systemic pro‐aging factor associated with function deficits in normal aging retinas. Heterochronic parabiosis and blood plasma treatment confirmed that systemic factors regulated age‐related inflammatory responses and the ectopic dendritic sprouting of rod bipolar (RBC) and horizontal (HC) cells in the aging retina. Proteomic analysis revealed that TPM1 was a potential systemic molecule underlying structural and functional deficits in the aging retina. Recombinant TPM1 protein administration accelerated the activation of glial cells, the dendritic sprouting of RBCs and HCs and functional decline in the retina of young mice, whereas anti‐TPM1 neutralizing antibody treatment ameliorated age‐related structural and function changes in the retina of aged mice. Old mouse plasma (OMP) induced glial cell activation and the dendritic outgrowth of RBCs and HCs in young mice, and yet TMP1‐depleted OMP failed to reproduce the similar effect in young mice. These results confirmed that TPM1 was a systemic pro‐aging factor. Moreover, we demonstrated that systematic TPM1 was an immune‐related molecule, which elicited endogenous TPM1 expression and inflammation by phosphorylating PKA and regulating FABP5/NF‐κB signaling pathway in normal aging retinas. Interestingly, we observed TPM1 upregulation and the ectopic dendritic sprouting of RBCs and HCs in young mouse models of Alzheimer's disease, indicating a potential role of TPM1 in age‐related neurodegenerative diseases. Our data indicate that TPM1 could be targeted for combating the aging process.

CqFlightless-I promotes the depolymerization of actin cytoskeleton to reduce white spot syndrome virus infection in Cherax quadricarinatus

As a member of the actin-associated protein gelsolin family, Flightless-I (FliI) is involved in promoting the depolymerization and rearrangement of the actin cytoskeleton, thus regulating a variety of biological processes, including cell proliferation, differentiation, apoptosis, and immune response. However, the molecular regulation of FliI is poorly understood in its regulation on viral infection in crustaceans. Previously, we found that the transcript of CqFliI was up-regulated in a differentially expressed transcriptome library of the hematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus at 1 h post white spot syndrome virus (WSSV) infection. To reveal the role of CqFliI in WSSV infection in crustacean, the open reading frame of CqFliI gene was cloned with 3870 bp, which encoded 1289 amino acids with eleven leucine-rich repeat (LRR) domains and six gelsolin-like (GEL) domains. Tissue distribution analysis showed that CqFliI was widely expressed in all the tested tissues with the highest expression in muscle, followed by a high expression in Hpt. In addition, the mRNA expression of CqFliI was significantly up-regulated in Hpt cells at 1, 3, 6, 12 and 24 h post WSSV infection. Importantly, the internalization of WSSV was clearly increased at an early infection stage of 1 hpi in Hpt cells by CqFliI gene silencing; meanwhile, the expression of viral immediate early gene IE1 and late viral envelope protein gene VP28 were both significantly increased after gene silencing of CqFliI in Hpt cells post WSSV infection, indicating a key role of CqFliI in reducing WSSV infection. Furthermore, protein pull-down assay revealed that CqFliI could bind with cytoskeletal β-actin through its GEL domains, rather than LRR domains; and the cytoskeleton of HeLa cells was obviously depolymerized after overexpression of GEL domain of CqFliI, implying that CqFliI might reduce the internalization of WSSV probably also the intracellular transport mediated by cytoskeleton via inhibiting actin polymerization. These findings may contribute to further understanding of WSSV infection mechanism which may provide more antiviral targeting design against WSSV disease.

Mechanical stress effects on transcriptional regulation of genes encoding microtubule- and actin-associated proteins

Plant cytoskeleton regulation has been studied using a new approach based on both (1) pharmacological analysis of tubulin and actin inhibitors and (2) mechanical stimulation achieved by using a slow-rotating (2 rpm) clinostat in combination with transcriptional analysis of genes encoding TUA6, ACT2, MAP65-1, CLASP, PLDδ, FH4 and FH1 proteins in Arabidopsis thaliana seedling roots. The obtained data suggest feedback between the organization of microtubule (MT) and actin filament (AF) networks and the expression of the ACT2, TUA6, MAP65-1, CLASP and FH1/FH4 genes. Different regulation of feedback between MT/AF organization and TUA6, ACT2, MAP65-1, CLASP, FH4 and FH1 gene expression was noted during slow clinorotation, possibly due to altered mechanical impact on the cortical cytoskeleton. For the first time, the expression of the tubulin-associated gene MAP65-1 was shown to be dependent upon the organization of AFs. TUA6, MAP65-1, CLASP, FH1 and FH4 likely participate in mechanical signal transduction. Our work demonstrated that slow clinorotation is able to cause mechanical stress.

Shroom3, a Gene Associated with CKD, Modulates Epithelial Recovery after AKI.

Ischemia-induced AKI resulting in tubular damage can often progress to CKD and is a common cause of nephrology consultation. After renal tubular epithelial damage, molecular and cellular mechanisms are activated to repair and regenerate the damaged epithelium. If these mechanisms are impaired, AKI can progress to CKD. Even in patients whose kidney function returns to normal baseline are more likely to develop CKD. Genome-wide association studies have provided robust evidence that genetic variants in Shroom3, which encodes an actin-associated protein, are associated with CKD and poor outcomes in transplanted kidneys. Here, we sought to further understand the associations of Shroom3 in CKD. Kidney ischemia was induced in wild-type (WT) and Shroom3 heterozygous null mice (Shroom3Gt/+ ) and the mechanisms of cellular recovery and repair were examined. A 28-minute bilateral ischemia in Shroom3Gt/+ mice resulted in 100% mortality within 24 hours. After 22-minute ischemic injury, Shroom3Gt/+ mice had a 16% increased mortality, worsened kidney function, and significantly worse histopathology, apoptosis, proliferation, inflammation, and fibrosis after injury. The cortical tubular damage in Shroom3Gt/+ was associated with disrupted epithelial redifferentiation, disrupted Rho-kinase/myosin signaling, and disorganized apical F-actin. Analysis of MDCK cells showed the levels of Shroom3 are directly correlated to apical organization of actin and actomyosin regulators. These findings establish that Shroom3 is required for epithelial repair and redifferentiation through the organization of actomyosin regulators, and could explain why genetic variants in Shroom3 are associated with CKD and allograft rejection.