scholarly journals Loss of Profilin3 Impairs Spermiogenesis by Affecting Acrosome Biogenesis, Autophagy, Manchette Development and Mitochondrial Organization

2021 ◽  
Vol 9 ◽  
Author(s):  
Naila Umer ◽  
Lena Arévalo ◽  
Sharang Phadke ◽  
Keerthika Lohanadan ◽  
Gregor Kirfel ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Regulatory Proteins ◽  
Autophagic Flux ◽  
Nuclear Fraction ◽  
Rna Seq ◽  
Head Shape ◽  
Male Factor ◽  
Protein Levels ◽  
Acrosome Formation ◽  
Actin Regulatory Proteins

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis, and metastasis of tumor cells. The testes-specific PFN3 is localized in the acroplaxome–manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3–/– males are subfertile, displaying a type II globozoospermia. We revealed that Pfn3–/– sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3–/– sperm showed reduced sperm motility resulting from flagellum deformities. We show that acrosome biogenesis is impaired starting from the Golgi phase, and mature sperm seems to suffer from a cytoplasm removal defect. An RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed, resulting in the inhibition of autophagy. This dysregulation of AMPK/mTOR affected the autophagic flux, which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3–/– testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3–ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3–/– testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicate this protein as a candidate for male factor infertility in humans.

Dynamin 2 (DNM2) and PACSIN2 Regulate Megakaryocyte Demarcation Membrane System Formation and Platelet Production in Concert

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 419-419
Author(s):  
Silvia Giannini ◽  
Markus Bender ◽  
Fred G. Pluthero ◽  
Hilary Christensen ◽  
Richard Leung ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Blood Platelets ◽  
Membrane System ◽  
Regulatory Proteins ◽  
Severe Thrombocytopenia ◽  
Platelet Production ◽  
Actin Regulatory Proteins ◽  
Dynamin 2

Abstract Blood platelets are produced in the bone marrow by megakaryocytes (MKs) in a process that requires extensive intracellular membrane rearrangements. These include the formation of the demarcation membrane system (DMS), the surface-connected membrane extension that invaginates into the cell body and further develops to provide membranes for future platelets. The precise molecular mechanisms responsible for these unique membrane rearrangements remain poorly understood. We have recently shown that Dnm2fl/fl Pf4-Cre mice specifically lacking the large GTPase dynamin 2 (DNM2) in MKs develop severe macrothrombocytopenia due to impaired receptor-mediated endocytosis (RME) (Bender, Giannini et al. Blood. 2015;125(6):1014-1024). Specifically, Dnm2fl/fl Pf4-Cre MKs accumulate arrested endocytic clathrin-coated vesicles that obstruct DMS formation. The actin nucleating factor Arp2/3 complex and polymerized actin clustered with clathrin at sites of impaired RME in Dnm2fl/fl Pf4-Cre MKs. We hypothesized that a DNM2 partner recruits actin-regulatory proteins at sites of RME and investigated the contribution of the F-BAR protein PACSIN2, an internal component of the initiating DMS (Jurak Begonja, Pluthero et al. Blood. 2015;126(1):80-88), in DMS formation and platelet production, as PACSIN2 interacts with DNM2 and actin-regulatory proteins such as N-WASP and filamin A (FlnA). Pacsin2-/- mice developed mild thrombocytopenia with slightly enlarged and shallow platelets. The DMS appeared less well defined and platelet territories were not readily visualized in Pacsin2-/- MKs. Pacsin2-/- Dnm2fl/fl Pf4-Cre mice lacking both PACSIN2 and DNM2 in MKs were further generated to determine the contribution of PACSIN2 in clathrin and actin clustering in Dnm2fl/fl Pf4-Cre MKs. Strikingly, PACSIN2 genetic deletion significantly improved the severe thrombocytopenia of Dnm2fl/fl Pf4-Cre mice. Specifically, PACSIN2 deletion abrogated the accumulation of clathrin and actin clusters, thereby unclogging DMS formation, which appeared as elongated maze-like membrane tubules in Pacsin2-/- Dnm2fl/fl Pf4-Cre MKs. Our results show that DNM2 terminates PACSIN2-dependent actin polymerization that accompanies RME, thereby allowing membrane rearrangements required for DMS formation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Analysis of Tetramethylrhodamine-labeled Actin Polymerization and Interaction with Actin Regulatory Proteins

2006 ◽  
Vol 281 (33) ◽  
pp. 24036-24047 ◽  
Author(s):  
Andrea Pelikan Conchaudron ◽  
Dominique Didry ◽  
Kim Ho Diep Le ◽  
Eric Larquet ◽  
Nicolas Boisset ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Regulatory Proteins ◽  
Actin Regulatory Proteins

scholarly journals Gelsolin-actin interaction and actin polymerization in human neutrophils.

1990 ◽  
Vol 110 (6) ◽  
pp. 1983-1991 ◽  
Author(s):  
T Howard ◽  
C Chaponnier ◽  
H Yin ◽  
T Stossel
Keyword(s):  
Human Blood ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Regulatory Proteins ◽  
Human Neutrophils ◽  
Blood Neutrophils ◽  
Acute Increase ◽  
Actin Regulatory Proteins ◽  
In Cells

The fraction of polymerized actin in human blood neutrophils increases after exposure to formyl-methionyl-leucyl-phenylalanine (fmlp), is maximal 10 s after peptide addition, and decreases after 300 s. Most of the gelsolin (85 +/- 11%) in resting ficoll-hypaque (FH)-purified neutrophils is in an EGTA resistant, 1:1 gelsolin-actin complex, and, within 5 s after 10(-7) M fmlp activation, the amount of gelsolin complexed with actin decreases to 42 +/- 12%. Reversal of gelsolin binding to actin occurs concurrently with an increase in F-actin content, and the appearance of barbed-end nucleating activity. The rate of dissociation of EGTA resistant, 1:1 gelsolin-actin complexes is more rapid in cells exposed to 10(-7) M fmlp than in cells exposed to 10(-9) M fmlp, and the extent of dissociation 10 s after activation depends upon the fmlp concentration. Furthermore, 300 s after fmlp activation when F-actin content is decreasing, gelsolin reassociates with actin as evidenced by an increase in the amount of EGTA resistant, 1:1 gelsolin-actin complex. Since fmlp induces barbed end actin polymerization in neutrophils and since in vitro the gelsolin-actin complex caps the barbed ends of actin filaments and blocks their growth, the data suggests that in FH neutrophils fmlp-induced actin polymerization could be initiated by the reversal of gelsolin binding to actin and the uncapping of actin filaments or nuclei. The data shows that formation and dissociation of gelsolin-actin complexes, together with the effects of other actin regulatory proteins, are important steps in the regulation of actin polymerization in neutrophils. Finally, finding increased amounts of gelsolin-actin complex in basal FH cells and dissociation of the complex in fmlp-activated cells suggests a mechanism by which fmlp can cause actin polymerization without an acute increase in cytosolic Ca++.

scholarly journals Bacterial effector targeting of a plant iron sensor facilitates iron acquisition and pathogen colonization

2021 ◽  
Author(s):  
Yingying Xing ◽  
Ning Xu ◽  
Deepak D Bhandari ◽  
Dmitry Lapin ◽  
Xinhua Sun ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Iron Uptake ◽  
Iron Acquisition ◽  
Iron Accumulation ◽  
Regulatory Proteins ◽  
Mineral Nutrient ◽  
Effector Protein ◽  
Iron Regulatory Proteins ◽  
Protein Levels ◽  
Pathogen Colonization

Abstract Acquisition of nutrients from different species is necessary for pathogen colonization. Iron is an essential mineral nutrient for nearly all organisms, but little is known about how pathogens manipulate plant hosts to acquire iron. Here, we report that AvrRps4, an effector protein delivered by Pseudomonas syringae bacteria to plants, interacts with and targets the plant iron sensor protein BRUTUS (BTS) to facilitate iron uptake and pathogen proliferation in Arabidopsis thaliana. Infection of rps4 and eds1 by P. syringae pv. tomato (Pst) DC3000 expressing AvrRps4 resulted in iron accumulation, especially in the plant apoplast. AvrRps4 alleviates BTS-mediated degradation of bHLH115 and ILR3(IAA-Leucine resistant 3), two iron regulatory proteins. In addition, BTS is important for accumulating immune proteins Enhanced Disease Susceptibility1 (EDS1) at both the transcriptional and protein levels upon Pst (avrRps4) infections. Our findings suggest that AvrRps4 targets BTS to facilitate iron accumulation and BTS contributes to RPS4/EDS1-mediated immune responses.

scholarly journals A new non-aggregative splicing isoform of human Tau is decreased in Alzheimer’s disease

2021 ◽  
Author(s):  
Vega García-Escudero ◽  
Daniel Ruiz-Gabarre ◽  
Ricardo Gargini ◽  
Mar Pérez ◽  
Esther García ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Neuroblastoma Cells ◽  
Rna Seq ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Post Translational Modifications ◽  
Protein Levels ◽  
Tau Isoform ◽  
Human Specific

AbstractTauopathies, including Alzheimer’s disease (AD) and frontotemporal lobar degeneration with Tau pathology (FTLD-tau), are a group of neurodegenerative disorders characterized by Tau hyperphosphorylation. Post-translational modifications of Tau such as phosphorylation and truncation have been demonstrated to be an essential step in the molecular pathogenesis of these tauopathies. In this work, we demonstrate the existence of a new, human-specific truncated form of Tau generated by intron 12 retention in human neuroblastoma cells and, to a higher extent, in human RNA brain samples, using qPCR and further confirming the results on a larger database of human RNA-seq samples. Diminished protein levels of this new Tau isoform are found by Westernblotting in Alzheimer’s patients’ brains (Braak I n = 3; Braak II n = 6, Braak III n = 3, Braak IV n = 1, and Braak V n = 10, Braak VI n = 8) with respect to non-demented control subjects (n = 9), suggesting that the lack of this truncated isoform may play an important role in the pathology. This new Tau isoform exhibits similar post-transcriptional modifications by phosphorylation and affinity for microtubule binding, but more interestingly, is less prone to aggregate than other Tau isoforms. Finally, we present evidence suggesting this new Tau isoform could be linked to the inhibition of GSK3β, which would mediate intron 12 retention by modulating the serine/arginine rich splicing factor 2 (SRSF2). Our results show the existence of an important new isoform of Tau and suggest that further research on this less aggregation-prone Tau may help to develop future therapies for Alzheimer’s disease and other tauopathies.

scholarly journals (Pro)renin receptor regulates autophagy and apoptosis in podocytes exposed to high glucose

2015 ◽  
Vol 309 (3) ◽  
pp. E302-E310 ◽  
Author(s):  
Caixia Li ◽  
Helmy M. Siragy
Keyword(s):  
Signaling Pathway ◽  
High Glucose ◽  
Caspase 3 ◽  
Mtor Signaling ◽  
Autophagic Flux ◽  
Bafilomycin A1 ◽  
Mtor Signaling Pathway ◽  
Autophagosome Formation ◽  
Podocyte Injury ◽  
Protein Levels

High glucose reduces autophagy and enhances apoptosis of podocytes. Previously, we reported that high glucose induced podocyte injury through upregulation of the (pro)renin receptor (PRR). We hypothesized that increasing PRR reduces autophagy and increases apoptosis of mouse podocytes exposed to high glucose via activation of the PI3K/Akt/mTOR signaling pathway. Mouse podocytes were cultured in normal (5 mmol/l) or high (25 mmol/l) d-glucose for 48 h. High glucose significantly increased mRNA and protein levels of PRR, phosphorylation of PI3K/Akt/mTOR, and p62. In contrast, high glucose decreased activation of UNC-51-like kinase-1 (ULK1) by phosphorylating Ser757 and protein levels of microtubule-associated protein-1 light chain 3B (LC3B)-II and Lamp-2. Bafilomycin A1 increased LC3BII and p62 accumulation in high-glucose-treated cells. High glucose reduced the autophagic flux. Confocal microscopy studies showed significant reduction in the protein level of LC3B in response to high glucose. Cyto-ID autophagy staining showed a significant decrease in autophagosome formation with high glucose. In the absence of PRR, activation of Akt with sc-79 or mTOR with MHY-1485 increased p62 accumulation. Caspase-3/7 activity and apoptosis monitored by TUNEL assay were significantly increased in podocytes treated with high glucose. PRR siRNA significantly reversed the effects of high glucose. Based on these data, we conclude that high glucose decreases autophagy and increases apoptosis in mouse podocytes through the PRR/PI3K/Akt/mTOR signaling pathway.

LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility

2011 ◽  
Vol 113 (1) ◽  
pp. 80-92 ◽  
Author(s):  
Galyna Kleveta ◽  
Kinga Borzęcka ◽  
Mykola Zdioruk ◽  
Maciej Czerkies ◽  
Hanna Kuberczyk ◽  
...  
Keyword(s):  
Regulatory Proteins ◽  
Actin Regulatory Proteins

scholarly journals miR-149 represses metastasis of hepatocellular carcinoma by targeting actin-regulatory proteins PPM1F

Oncotarget ◽  
2015 ◽  
Vol 6 (35) ◽  
pp. 37808-37823 ◽  
Author(s):  
Gang Luo ◽  
Ya-Ling Chao ◽  
Bo Tang ◽  
Bo-Sheng Li ◽  
Yu-Feng Xiao ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Regulatory Proteins ◽  
Actin Regulatory Proteins

scholarly journals Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Nicolò Tosetti ◽  
Nicolas Dos Santos Pacheco ◽  
Dominique Soldati-Favre ◽  
Damien Jacot
Keyword(s):  
Protein Kinase ◽  
Toxoplasma Gondii ◽  
Actin Polymerization ◽  
Cell Communication ◽  
Residual Body ◽  
Filamentous Actin ◽  
Calcium Dependent ◽  
Actin Regulatory Proteins ◽  
Parasite Motility ◽  
Cell Cell

Toxoplasma gondii possesses a limited set of actin-regulatory proteins and relies on only three formins (FRMs) to nucleate and polymerize actin. We combined filamentous actin (F-actin) chromobodies with gene disruption to assign specific populations of actin filaments to individual formins. FRM2 localizes to the apical juxtanuclear region and participates in apicoplast inheritance. Restricted to the residual body, FRM3 maintains the intravacuolar cell-cell communication. Conoidal FRM1 initiates a flux of F-actin crucial for motility, invasion and egress. This flux depends on myosins A and H and is controlled by phosphorylation via PKG (protein kinase G) and CDPK1 (calcium-dependent protein kinase 1) and by methylation via AKMT (apical lysine methyltransferase). This flux is independent of microneme secretion and persists in the absence of the glideosome-associated connector (GAC). This study offers a coherent model of the key players controlling actin polymerization, stressing the importance of well-timed post-translational modifications to power parasite motility.

scholarly journals Chorionic villus-derived mesenchymal stem cell-mediated autophagy promotes proliferation and invasiveness of trophoblasts under hypoxia by activating the JAK2/STAT3 signalling pathway

2020 ◽  
Author(s):  
Yijing Chu ◽  
Chongyu Yue ◽  
Wei Peng ◽  
Weiping Chen ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chorionic Villus ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Upstream Regulator ◽  
Pathway Analyses ◽  
Stat3 Signalling

Abstract Objectives Trophoblast dysfunction during pregnancy is fundamentally involved in preeclampsia. The aim of this study was to understand how human chorionic villous mesenchymal stem cells (CV-MSCs) operate in regulation of trophoblast function.Materials and Methods We treated trophoblasts with CV-MSC supernatant under hypoxic conditions, and transcriptome and pathway analyses of trophoblasts were performed. Western blotting and PCR analysis were used to examine the JAK2, STAT3 and autophagy associated protein expression levels in trophoblasts.Results The CV-MSC supernatant treatment markedly enhanced proliferation, invasion and autophagy. The RNA-seq revealed JAK2/STAT3 signalling as an upstream regulator, and STAT3 mRNA and protein levels increased during CV-MSC treatment. Inhibition of JAK2/STAT3 signalling reduced autophagy, survival and invasion of trophoblasts even in the presence of CV-MSCs, and blocking autophagy did not affect STAT3 activation in trophoblasts treated with CV-MSCs. Importantly, overexpression of STAT3 increased the levels of autophagy in trophoblasts; thus, it regulated positively autophagy in hypoxic trophoblasts. Human placental explants also proved our finding, in which STAT3 was activated and LC3B-II levels were increased by CV-MSC treatment.Conclusions Our data suggest that CV-MSC-dependent activation of JAK2/STAT3 signalling is a prerequisite for upregulation of autophagy in trophoblasts.

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