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BioRxiv : the Preprint Server For... Jun 2024Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the...
Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of , a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.
PubMed: 38915624
DOI: 10.1101/2024.06.12.598725 -
BioRxiv : the Preprint Server For... Jun 2024Inverted formin-2 (INF2) gene mutations are among the most common causes of genetic focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth (CMT)...
Inverted formin-2 (INF2) gene mutations are among the most common causes of genetic focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth (CMT) disease. Recent studies suggest that INF2, through its effects on actin and microtubule arrangement, can regulate processes including vesicle trafficking, cell adhesion, mitochondrial calcium uptake, mitochondrial fission, and T-cell polarization. Despite roles for INF2 in multiple cellular processes, neither the human pathogenic R218Q INF2 point mutation nor the INF2 knock-out allele is sufficient to cause disease in mice. This discrepancy challenges our efforts to explain the disease mechanism, as the link between INF2-related processes, podocyte structure, disease inheritance pattern, and their clinical presentation remains enigmatic. Here, we compared the kidney responses to puromycin aminonucleoside (PAN) induced injury between R218Q INF2 point mutant knock-in and INF2 knock-out mouse models and show that R218Q INF2 mice are susceptible to developing proteinuria and FSGS. This contrasts with INF2 knock-out mice, which show only a minimal kidney phenotype. Co-localization and co-immunoprecipitation analysis of wild-type and mutant INF2 coupled with measurements of cellular actin content revealed that the R218Q INF2 point mutation confers a gain-of-function effect by altering the actin cytoskeleton, facilitated in part by alterations in INF2 localization. Differential analysis of RNA expression in PAN-stressed heterozygous R218Q INF2 point-mutant and heterozygous INF2 knock-out mouse glomeruli showed that the adhesion and mitochondria-related pathways were significantly enriched in the disease condition. Mouse podocytes with R218Q INF2, and an INF2-mutant human patient's kidney organoid-derived podocytes with an S186P INF2 mutation, recapitulate the defective adhesion and mitochondria phenotypes. These results link INF2-regulated cellular processes to the onset and progression of glomerular disease. Thus, our data demonstrate that gain-of-function mechanisms drive INF2-related FSGS and explain the autosomal dominant inheritance pattern of this disease.
PubMed: 38915495
DOI: 10.1101/2024.06.08.598088 -
PLoS Genetics Jun 2024Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin...
Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin filaments from opposing sarcomeres, the smallest contractile units of muscles. This happens at the Z-disc, the actin-organizing center of sarcomeres. In flies and vertebrates, filamin mutations lead to fragile muscles that appear ruptured, suggesting filamin helps counteract muscle rupturing during muscle contractions by providing elastic support and/or through signaling. An elastic region at the C-terminus of filamin is called the mechanosensitive region and has been proposed to sense and counteract contractile damage. Here we use molecularly defined mutants and microscopy analysis of the Drosophila indirect flight muscles to investigate the molecular details by which filamin provides cohesion to the Z-disc. We made novel filamin mutations affecting the C-terminal region to interrogate the mechanosensitive region and detected three Z-disc phenotypes: dissociation of actin filaments, Z-disc rupture, and Z-disc enlargement. We tested a constitutively closed filamin mutant, which prevents the elastic changes in the mechanosensitive region and results in ruptured Z-discs, and a constitutively open mutant which has the opposite elastic effect on the mechanosensitive region and gives rise to enlarged Z-discs. Finally, we show that muscle contraction is required for Z-disc rupture. We propose that filamin senses myofibril damage by elastic changes in its mechanosensory region, stabilizes the Z-disc, and counteracts contractile damage at the Z-disc.
PubMed: 38905299
DOI: 10.1371/journal.pgen.1011101 -
Frontiers in Bioengineering and... 2024The stiffness of the extracellular matrix plays a crucial role in cell motility and spreading, influencing cell morphology through cytoskeleton organization and...
The stiffness of the extracellular matrix plays a crucial role in cell motility and spreading, influencing cell morphology through cytoskeleton organization and transmembrane proteins' expression. In this context, mechanical characterization of both cells and the extracellular matrix gains prominence for enhanced diagnostics and clinical decision-making. Here, we investigate the combined effect of mechanotransduction and ionizing radiations on altering cells' mechanical properties, analysing mammary cell lines (MCF10A and MDA-MB-231) after X-ray radiotherapy (2 and 10 Gy). We found that ionizing radiations sensitively affect adenocarcinoma cells cultured on substrates mimicking cancerous tissue stiffness (15 kPa), inducing an increased structuration of paxillin-rich focal adhesions and cytoskeleton: this process translates in the augmentation of tension at the actin filaments level, causing cellular stiffness and consequently affecting cytoplasmatic/nuclear morphologies. Deeper exploration of the intricate interplay between mechanical factors and radiation should provide novel strategies to orient clinical outcomes.
PubMed: 38903185
DOI: 10.3389/fbioe.2024.1408789 -
BioRxiv : the Preprint Server For... Mar 2024The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC...
The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine F-actin structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of Mrtf, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G-protein-coupled receptor (GPCR) Smog, G-protein αq subunit, Rho1 GTPase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand Fog to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, a NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.
PubMed: 38903085
DOI: 10.1101/2024.03.11.584337 -
Molecular Pharmacology Jun 2024Transmembrane signaling is a critical process by which changes in the extracellular environment are relayed to intracellular systems that induce changes in homeostasis....
Transmembrane signaling is a critical process by which changes in the extracellular environment are relayed to intracellular systems that induce changes in homeostasis. One common intracellular system involves guanine nucleotide exchange factors (GEFs), which catalyzes the exchange of GTP for GDP bound to inactive guanine nucleotide binding proteins (G proteins). The resulting active G proteins then interact with downstream targets that control cell proliferation, growth, shape, migration, adhesion, and transcription. Dysregulation of any of these processes is a hallmark of cancer. The Dbl family of GEFs activate Rho family G proteins, which in turn alter the actin cytoskeleton and promote gene transcription. Although they have a common catalytic mechanism exercised by their conserved Dbl homology (DH) domains, Dbl GEFs are regulated in very diverse ways. Often, this regulation involves the release of autoinhibition imposed by accessory domains. Amongst these domains, the pleckstrin homology (PH) domain is the most conserved and is almost always found immediately C-terminal to the DH domain. The domain been associated with both positive and negative regulation. Recently, some atomic structures of Dbl GEFs have been determined which reemphasize the complex and central role that the PH domain can play in orchestrating regulation of the DH domain. Here we discuss these newer structures, put them into context by cataloging the various ways that PH domains are known to contribute to signaling across the Dbl family, and discuss how the PH might be exploited to achieve selective inhibition of this protein family by small molecule therapeutics. Dysregulation via overexpression or mutation of Dbl family RhoGEFs contributes disease. Targeting the Dbl homology (DH) catalytic domain by small molecule therapeutics has been challenging due to its high conservation and the lack of a discrete binding pocket. By evaluating some new autoinhibitory mechanisms in the Dbl family, we demonstrate the great diversity of roles played by the regulatory domains, in particular the PH domain, and how this holds tremendous potential for development of selective therapeutics that modulate GEF activity.
PubMed: 38902036
DOI: 10.1124/molpharm.124.000904 -
Acta Tropica Jun 2024Soluble factors in the secretome of Acanthamoeba castellanii play crucial roles in the pathogenesis of Acanthamoeba keratitis (AK). Investigating the pathological...
Soluble factors in the secretome of Acanthamoeba castellanii play crucial roles in the pathogenesis of Acanthamoeba keratitis (AK). Investigating the pathological effects of A. castellanii-derived conditioned medium (ACCM) on ocular cells can provide insights into the damage inflicted during AK. This study examined ACCM-induced cytotoxicity in primary human corneal stromal cells (CSCs) and a human SV40 immortalized corneal epithelial cell line (ihCECs) at varying ACCM concentrations (25 %, 50 %, 75 %, and 100 %). MTT, AlamarBlue, Sulforhodamine B, lactate dehydrogenase, and Caspase-3/7 activation assays were used to assess the impact of ACCM on the cell viability, proliferation and apoptosis. Additionally, fluorescent staining was used to reveal actin cytoskeleton changes. ACCM exposure significantly decreased cell viability, increased apoptosis, and disrupted the actin cytoskeleton, particularly at higher concentrations and longer exposures. Proteases were found to mediate these cytopathogenic effects, highlighting the need for characterization of A. castellanii proteases as key virulence factors in AK pathogenesis.
PubMed: 38901524
DOI: 10.1016/j.actatropica.2024.107288 -
BioRxiv : the Preprint Server For... Jun 2024The mechanical properties of the mammalian cell regulate many cellular functions and are largely dictated by the cytoskeleton, a composite network of protein filaments,...
The mechanical properties of the mammalian cell regulate many cellular functions and are largely dictated by the cytoskeleton, a composite network of protein filaments, including actin, microtubules, and intermediate filaments. Interactions between these distinct filaments give rise to emergent mechanical properties that are difficult to generate synthetically, and recent studies have made great strides in advancing our understanding of the mechanical interplay between actin and microtubule filaments. While intermediate filaments play critical roles in the stress response of cells, their effect on the rheological properties of the composite cytoskeleton remains poorly understood. Here, we use optical tweezers microrheology to measure the linear viscoelastic properties and nonlinear stress response of composites of actin and vimentin with varying molar ratios of actin to vimentin. We reveal a surprising, nearly opposite effect of actin-vimentin network mechanics compared to single-component networks in the linear versus nonlinear regimes. Namely, the linear elastic plateau modulus and zero-shear viscosity are markedly reduced in composites compared to single-component networks of actin or vimentin, whereas the initial response force and stiffness are maximized in composites versus single-component networks in the nonlinear regime. While these emergent trends are indicative of distinct interactions between actin and vimentin, nonlinear stiffening and longtime stress response appear to both be dictated primarily by actin, at odds with previous bulk rheology studies. We demonstrate that these complex, scale-dependent effects arise from the varied contributions of network density, filament stiffness, non-specific interactions, and poroelasticity to the mechanical response at different spatiotemporal scales. Cells may harness this complex behavior to facilitate distinct stress responses at different scales and in response to different stimuli to allow for their hallmark multifunctionality.
PubMed: 38895280
DOI: 10.1101/2024.06.07.597906 -
F1000Research 2022Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has...
BACKGROUND
Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC).
METHODS
Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors.
RESULTS
Fascin immunopositivity was observed in peripheral ameloblast-like cells, and weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which is an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported.
CONCLUSIONS
Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.
Topics: Humans; Transcription Factors; Microfilament Proteins; Odontogenic Cysts; Carrier Proteins; Immunohistochemistry; Ameloblastoma; Odontogenic Tumors; Biomarkers, Tumor
PubMed: 38895097
DOI: 10.12688/f1000research.126091.3 -
International Journal of Molecular... Jun 2024Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disease at risk of sudden death. Genetic testing impacts greatly in ACM diagnosis, but gene-disease...
Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disease at risk of sudden death. Genetic testing impacts greatly in ACM diagnosis, but gene-disease associations have yet to be determined for the increasing number of genes included in clinical panels. Genetic variants evaluation was undertaken for the most relevant non-desmosomal disease genes. We retrospectively studied 320 unrelated Italian ACM patients, including 243 cases with predominant right-ventricular (ARVC) and 77 cases with predominant left-ventricular (ALVC) involvement, who did not carry pathogenic/likely pathogenic (P/LP) variants in desmosome-coding genes. The aim was to assess rare genetic variants in transmembrane protein 43 (), desmin (), phospholamban (), filamin c (), cadherin 2 (), and tight junction protein 1 (), based on current adjudication guidelines and reappraisal on reported literature data. Thirty-five rare genetic variants, including 23 (64%) P/LP, were identified in 39 patients (16/243 ARVC; 23/77 ALVC): 22 , 9 , 2 , and 2 . No P/LP variants were found in and genes. Gene-based burden analysis, including P/LP variants reported in literature, showed significant enrichment for (3.79-fold), (10.31-fold), (117.8-fold) and (107-fold). A non-desmosomal rare genetic variant is found in a minority of ARVC patients but in about one third of ALVC patients; as such, clinical decision-making should be driven by genes with robust evidence. More than two thirds of non-desmosomal P/LP variants occur in FLNC.
Topics: Humans; Arrhythmogenic Right Ventricular Dysplasia; Female; Male; Adult; Middle Aged; Membrane Proteins; Cadherins; Desmosomes; Genetic Predisposition to Disease; Genetic Variation; Filamins; Retrospective Studies; Italy; Calcium-Binding Proteins; Antigens, CD
PubMed: 38892455
DOI: 10.3390/ijms25116267