Analysis of Tetramethylrhodamine-labeled Actin Polymerization and Interaction with 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.

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

Blood ◽

10.1182/blood.v126.23.419.419 ◽

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.

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Gelsolin-actin interaction and actin polymerization in human neutrophils.

The Journal of Cell Biology ◽

10.1083/jcb.110.6.1983 ◽

1990 ◽

Vol 110 (6) ◽

pp. 1983-1991 ◽

Cited By ~ 38

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++.

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

Journal of Cellular Biochemistry ◽

10.1002/jcb.23330 ◽

2011 ◽

Vol 113 (1) ◽

pp. 80-92 ◽

Cited By ~ 31

Author(s):

Galyna Kleveta ◽

Kinga Borzęcka ◽

Mykola Zdioruk ◽

Maciej Czerkies ◽

Hanna Kuberczyk ◽

...

Keyword(s):

Regulatory Proteins ◽

Actin Regulatory Proteins

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

Oncotarget ◽

10.18632/oncotarget.5676 ◽

2015 ◽

Vol 6 (35) ◽

pp. 37808-37823 ◽

Cited By ~ 36

Author(s):

Gang Luo ◽

Ya-Ling Chao ◽

Bo Tang ◽

Bo-Sheng Li ◽

Yu-Feng Xiao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Regulatory Proteins ◽

Actin Regulatory Proteins

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

eLife ◽

10.7554/elife.42669 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 27

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.

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