Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

A Novel In Silico Method for Molecular Mimicry Detection Finds a Formin with the Potential to Manipulate the Maize Cell Cytoskeleton.

Molecular mimicry is one of the evolutionary strategies that parasites use to manipulate the host metabolism and perform an effective infection. This phenomenon has been observed in several animal and plant pathosystems. Despite the relevance of this mechanism in pathogenesis, little is known about it in fungus-plant interactions. For that reason, we performed an in silico method to select plausible mimicry candidates for the Ustilago maydis-maize interaction. Our methodology used a tripartite sequence comparison between the parasite, the host, and nonparasitic organisms' genomes. Furthermore, we used RNA sequencing information to identify gene coexpression, and we determined subcellular localization to detect potential cases of colocalization in the imitator-imitated pairs. With these approximations, we found a putative extracellular formin in U. maydis with the potential to rearrange the host cell cytoskeleton. In parallel, we detected at least two maize genes involved in the cytoskeleton rearrangement differentially expressed under U. maydis infection; thus, this find increases the expectation for the potential mimicry role of the fungal protein. The use of several sources of data led us to develop a strict and replicable in silico methodology to detect molecular mimicry in pathosystems with enough information available. Furthermore, this is the first time that a genomewide search has been performed to detect molecular mimicry in a U. maydis-maize system. Additionally, to allow the reproducibility of this experiment and the use of this pipeline, we created a Web server called Molecular Mimicry Finder.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Following the footprints of variability during filopodial growth.

Filopodia are actin-built finger-like dynamic structures that protrude from the cell cortex. These structures can sense the environment and play key roles in migration and cell-cell interactions. The growth-retraction cycle of filopodia is a complex process exquisitely regulated by intra- and extra-cellular cues, whose nature remains elusive. Filopodia present wide variation in length, lifetime and growth rate. Here, we investigate the features of filopodia patterns in fixed prostate tumor cells by confocal microscopy. Analysis of almost a thousand filopodia suggests the presence of two different populations: one characterized by a narrow distribution of lengths and the other with a much more variable pattern with very long filopodia. We explore a stochastic model of filopodial growth which takes into account diffusion and reactions involving actin and the regulatory proteins formin and capping, and retrograde flow. Interestingly, we found an inverse dependence between the filopodial length and the retrograde velocity. This result led us to propose that variations in the retrograde velocity could explain the experimental lengths observed for these tumor cells. In this sense, one population involves a wider range of retrograde velocities than the other population, and also includes low values of this velocity. It has been hypothesized that cells would be able to regulate retrograde flow as a mechanism to control filopodial length. Thus, we propound that the experimental filopodia pattern is the result of differential retrograde velocities originated from heterogeneous signaling due to cell-substrate interactions or prior cell-cell contacts.

FHOD3 promotes carcinogenesis by regulating RhoA/ROCK1/LIMK1 signaling pathway in medulloblastoma.

PURPOSE: Medulloblastoma (MB) is a malignant brain disease in young children. The overall survival of MB patients is disappointing due to absence of effective therapeutics and this could be attributed to the lack of molecular mechanism underlying MB. FHOD3 was an important gene during cardio-genesis and was reported to promote cell migration in cancer. However, its role in MB is not clear to date. METHODS: RT-qPCR and IHC analysis were used to determine expression of FHOD3. Survival curve was drawn by K-M analysis. FHOD3 was knocked down by RNAi technology. The effects of FHOD3 on medulloblastoma cells were determined by CCK-8 assay, colony formation assay, transwell assay and FACs analysis. RESULTS: FHOD3 expression increased by 1.5 fold in tumor tissues compared to the control and IHC analysis further confirmed strong expression of FHOD3 in medulloblastoma tissues. Then higher FHOD3 expression was associated with shorter survival time in MB patients (13.0 months versus 43.8 months). In medulloblastoma cells such as Daoy and D283med, FHOD3 also displayed abundant expression. When FHOD3 was knocked down, the ability of cell proliferation and colony formation was reduced over greatly. The capability of cell migration and invasion was also inhibited significantly. However, cell apoptotic rate increased significantly reversely. Mechanistically, the phosphorylation level of RhoA, ROCK1, and LIMK1 was decreased when FHOD3 was knocked down but increased reversely when FHOD3 was over-expressed in Daoy cells. CONCLUSIONS: FHOD3 was associated with overall survival time in medulloblastoma patients and was essential to cell proliferation, growth and survival in medulloblastoma and might regulates activation of RhoA/ROCK1/LIMK1 signaling pathway.

TNF induces neutrophil adhesion via formin-dependent cytoskeletal reorganization and activation of ß-integrin function.

Although essential for inflammatory responses, leukocyte recruitment to blood vessel walls in response to inflammatory stimuli, such as TNF-α, can contribute to vascular occlusion in inflammatory diseases, including atherosclerosis. We aimed to further characterize the mechanisms by which TNF stimulates adhesive and morphologic alterations in neutrophils. Microfluidic and intravital assays confirmed the potent effect that TNF has on human and murine neutrophil adhesion and recruitment in vitro and in vivo, respectively. Inhibition of actin polymerization by cytochalasin D significantly diminished TNF-induced human neutrophil adhesion in vitro and abolished TNF-induced membrane alterations and cell spreading. In contrast, TNF-induced increases in ß2-integrin (Mac-1 and LFA-1) expression was not significantly altered by actin polymerization inhibition. Consistent with a role for cytoskeletal rearrangements in TNF-induced adhesion, TNF augmented the activity of the Rho GTPase, RhoA, in human neutrophils. However, inhibition of the major RhoA effector protein, Rho kinase (ROCK), by Y-27632 failed to inhibit TNF-induced neutrophil adhesion. In contrast, the formin FH2 domain inhibitor, SMIFH2, abolished TNF-induced human neutrophil adhesion and diminished leukocyte recruitment in vivo. SMIFH2 also inhibited TNF-induced cytoskeletal reorganization in human neutrophils and abolished the alterations in ß2-integrin expression elicited by TNF stimulation. As such, Rho GTPase/mDia formin-mediated cytoskeletal reorganization appears to participate in the orchestration of TNF-induced neutrophil-adhesive interactions, possibly mediated by formin-mediated actin nucleation and subsequent modulation of ß2-integrin activity on the neutrophil surface. This pathway may represent a pharmacologic target for reducing leukocyte recruitment in inflammatory diseases.

Effect of miR-29c and miR-129-5p on epithelial-mesenchymal transition in experimental biliary atresia mouse models.

Biliary atresia (BA) is a destructive bile duct disease occurring in newborn children within a few weeks after birth. In this study, the effect of miR-29c and miR-129-5p on epithelial-mesenchymal transition (EMT) in experimental BA was explored by constructing BA mouse models via Rhesus rotavirus vaccine infection. miR-29c and miR-129-5p expression was analyzed by real-time quantitative polymerase chain reaction. EMT was established by induction with transforming growth factor (TGF)-ß1. miR-29c and miR-129-5p were overexpressed and inhibited, respectively, by Lipofectamine transfection. EMT-related protein (formin-like 2, FMNL2; E-cadherin; vimentin; and cytokeratin-19, CK-19) expression was analyzed by western blot and immunofluorescent assay. The results indicated that miR-29c and miR-129-5p were downregulated and upregulated in BA mice. TGF-ß1 induction caused a time-dependent decrease and increase in miR-29c and miR-129-5p, respectively. Additionally, TGF-ß1 induced an increase in FMNL2 and vimentin expression and a decrease in E-cadherin and CK-19 expression (P < 0.05). Overexpression or suppression of miRNA-29c or miR-129-5p, respectively, induced the inhibition of FMNL2 and vimentin, and promotion of E-cadherin and CK-19 expression, in the test groups compared to the non-intervention group (P < 0.05). However, the FMNL2, vimentin, E-cadherin, and CK- 19 expression did not differ between the control and non-intervention groups (P > 0.05). Thus, miR-29c upregulation or miR-129-5p downregulation effectively prevented EMT in BA by regulating the expression of EMT pathway-related proteins. Therefore, miR-29c and miR-129-5p could be utilized as therapeutic targets for BA in the future.

Lethal Disorder of Mitochondrial Fission Caused by Mutations in DNM1L.

We describe two infants with hypotonia, absent respiratory effort, and giant mitochondria in neurons due to compound heterozygosity for 2 nonsense mutations of DNM1L. DNM1L has a critical role in regulating mitochondrial morphology and function. This observation confirms the central role of mitochondrial fission to normal human development.

An inherited small microdeletion at 15q13.3 in a patient with early-onset obsessive-compulsive disorder.

Copy number variations (CNVs) have been previously associated with several different neurodevelopmental psychiatric disorders, such as autism, schizophrenia, and attention deficit hyperactivity disorder (ADHD). The present study consisted of a pilot genome-wide screen for CNVs in a cohort of 16 patients with early-onset obsessive-compulsive disorder (OCD) and 12 mentally healthy individuals, using array-based comparative genomic hybridization (aCGH) on 44K arrays. A small rare paternal inherited microdeletion (∼64 kb) was identified in chromosome 15q13.3 of one male patient with very early onset OCD. The father did not have OCD. The deletion encompassed part of the FMN1 gene, which is involved with the glutamatergic system. This finding supports the hypothesis of a complex network of several genes expressed in the brain contributing for the genetic risk of OCD, and also supports the glutamatergic involvement in OCD, which has been previously reported in the literature.

FMNL1 promotes proliferation and migration of leukemia cells.

The human FMNL1 is expressed predominantly in hematopoietic cells and has been described previously as overexpressed in hematopoietic malignancies. However, it is not known whether FMNL1 contributes to leukemogenesis. Here, we investigate the FMNL1 function using two different human leukemia models: Namalwa and K562 cell lines. FMNL1 depletion reduced cell proliferation and colony formation in both leukemic cell types, as well as a decrease in the tumor growth of FMNL1-depleted Namalwa cell xenografts. In addition, there was a decrease in migration and in TEM in FMNL1-depleted Namalwa cells. FMNL1 endogenously associates with Rac1, and FMNL1 silencing resulted in an increased Rac1 activity. The reduced migration observed in FMNL1-depleted cells was restored by inhibiting Rac activity. Our results indicate that FMNL1 stimulates leukemia cell proliferation as well as migration. This suggests that FMNL1 contributes to leukemogenesis and could act in part through Rac1 regulation.

Fhos encodes a Drosophila Formin-like protein participating in autophagic programmed cell death.

Larval tissues undergo programmed cell death (PCD) during Drosophila metamorphosis. PCD is triggered in a stage and tissue-specific fashion in response to ecdysone pulses. The understanding of how ecdysone induces the stage and tissue-specificity of cell death remains obscure. Several steroid-regulated primary response genes have been shown to act as key regulators of cellular responses to ecdysone by inducing a cascade of transcriptional regulation of late responsive genes. In this article, the authors identify Fhos as a gene that is required for Drosophila larval salivary gland destruction. Animals with a P-element mutation in Fhos possess persistent larval salivary glands, and precise excisions of this P-element insertion resulted in reversion of this salivary gland mutant phenotype. Fhos encodes the Drosophila homolog of mammalian Formin Fhos. Fhos is differentially transcribed during development and responds to ecdysone in a method that is similar to other cell death genes. Similarly to what has been shown for its mammalian counterpart, FHOS protein is translocated to the nucleus at later stages of cell death. Fhos mutants posses disrupted actin cytoskeleton dynamics in persistent salivary glands. Together, our data indicate that Fhos is a new ecdysone-regulated gene that is crucial for changes in the actin cytoskeleton during salivary gland elimination in Drosophila.

Structural basis for APPTPPPLPP peptide recognition by the FBP11WW1 domain.

WW domains are small protein-protein interaction modules that recognize proline-rich stretches in proteins. The class II tandem WW domains of the formin binding protein 11 (FBP11) recognize specifically proteins containing PPLPp motifs as present in the formins that are involved in limb and kidney development, and in the methyl-CpG-binding protein 2 (MeCP2), associated with the Rett syndrome. The interaction involves the specific recognition of a leucine side-chain. Here, we report on the novel structure of the complex formed by the FPB11WW1 domain and the formin fragment APPTPPPLPP revealing the specificity determinants of class II WW domains.

Human leukocyte formin: a novel protein expressed in lymphoid malignancies and associated with Akt.

The very large family of Formin proteins is involved in processes such as morphogenesis, embryonic differentiation, cell polarity, and cytokinesis. A novel human gene from the Formin family, denominated human leukocyte formin gene, was cloned. The cDNA of the gene was determined to be 3959bp long with an open reading frame of 3302bp and computational analysis located this gene on chromosome 17, suggesting that it is composed of 27 exons. Northern blot analysis revealed a restricted expression of mRNA in the thymus, spleen, and peripheral blood leukocytes in normal human tissues. Western blot analysis demonstrated that the protein encoded by this gene is overexpressed in lymphoid malignancies; cancer cell lines and peripheral blood leukocyte from chronic lymphocytic leukemia (CLL) patients. Furthermore, the human leukocyte formin protein was observed to associate with Akt, a critical survival regulator in many different cell types.

Further characterization of the DFNA1 audiovestibular phenotype.

BACKGROUND: Autosomal dominant, nonsyndromic, hereditary hearing impairment in a large Costa Rican kindred is caused by a mutation in the human homolog of the Drosophila diaphanous gene. OBJECTIVE: To further characterize the phenotype of DFNA1 with comprehensive audiovestibular evaluation and computed tomography of the temporal bone. PATIENTS: One affected child and 2 affected adults of the Costa Rican kindred who harbor a mutation in the diaphanous gene. SETTING: Medical Center at the University of California, San Francisco. INTERVENTION: Otologic and neuro-otologic examination; pure tone audiometry, speech audiometry, and immitance testing; auditory evoked potentials, electrocochleography, and otoacoustic emissions; electronystagmography and vestibular autorotation tests; and computed tomography of the temporal bone. RESULTS: The youngest subject, an 8-year-old boy, had a mild hearing loss, intact stapedial reflexes, otoacoustic emissions at high frequencies, normal auditory evoked potentials, and electrocochleographic findings consistent with endolymphatic hydrops. The two adults had severe to profound bilateral sensorineural hearing impairment. Electronystagmography disclosed normal vestibular function. Computed tomography demonstrated normal external, middle, and inner ear structures. CONCLUSIONS: These results suggest that the early low-frequency hearing loss in this family is associated with endolymphatic hydrops. Elucidation of the role of the diaphanous gene in hearing will therefore lead to a better understanding of the mechanism of endolymphatic hydrops.