Dual regulation of the actin cytoskeleton by CARMIL-GAP

ABSTRACTCARMIL (Capping proteinArp2/3MyosinI Linker) proteins are multi-domain scaffold proteins that regulate actin dynamics by regulating the activity of Capping Protein (CP). Here we characterize CARMIL-GAP, aDictyosteliumCARMIL isoform that contains a ~130 residue insert that, by homology, is a GTPase activating (GAP) domain for Rho-related GTPases. Consistently, this GAP domain bindsDictyosteliumRac1a and accelerates its rate of GTP hydrolysis. CARMIL-GAP concentrates with F-actin in phagocytic cups and at the leading edge of chemotaxing cells, and cells devoid of CARMIL-GAP exhibit pronounced defects in phagocytosis and chemotactic streaming. Importantly, these defects are fully rescued by the re-expression of CARMIL-GAP. Finally, the rescue of CARMIL-GAP null cells with versions of CARMIL-GAP that lack either GAP activity or the ability to regulate CP show that while both activities contribute significantly to CARMIL-GAP function, the GAP activity plays the bigger role. Together, our results add to the growing evidence that CARMIL proteins influence actin dynamics by regulating signaling molecules as well as CP, and that the continuous cycling of Rho GTPases between their GTP and GDP bound states is often required to drive Rho-dependent biological processes.SUMMARY STATEMENTThe assembly of actin filaments supports a wide array of fundamental cellular functions, including cell migration and phagocytosis. Actin assembly is controlled by a host of regulatory proteins, with Capping Protein being one of the most important. Capping Protein is in turn regulated by the CARMIL family of proteins. Actin assembly is also controlled by signaling pathways that often converge on Rho-related GTPases like Rac1. These GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. Guanine nucleotide exchange factors (GEFs) and guanine nucleotide activating proteins (GAPs) drive Rho-related GTPases to their GTP-bound and GDP-bound states, respectively. Here we characterized a version of CARMIL that contains within it a GAP domain for Rac1. We show that CARMILGAP supports the actin-based processes of cell migration and phagocytosis. We also show that while CARMIL-GAP’s ability to regulate Capping Protein and the nucleotide state of Rac1 are both important for its cellular functions, its ability to regulate Rac1 via its GAP domain plays the bigger role. Finally, our data support the emerging concept that the continuous cycling of Rho GTPases between their GTP-bound and GDP-bound states is often required to drive Rho-dependent biological processes.

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Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization in Drosophila

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0103 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3107-3116 ◽

Cited By ~ 29

Author(s):

Anindya Ghosh-Roy ◽

Bela S. Desai ◽

Krishanu Ray

Keyword(s):

Light Chain ◽

Cytoplasmic Dynein ◽

Actin Dynamics ◽

Dynein Light Chain ◽

Actin Assembly ◽

Cellular Functions ◽

Directed Movement ◽

Cellular Processes ◽

Dynactin Complex

Toward the end of spermiogenesis, spermatid nuclei are compacted and the clonally related spermatids individualize to become mature and active sperm. Studies in Drosophila showed that caudal end-directed movement of a microfilament-rich structure, called investment cone, expels the cytoplasmic contents of individual spermatids. F-actin dynamics plays an important role in this process. Here we report that the dynein light chain 1 (DLC1) of Drosophila is involved in two separate cellular processes during sperm individualization. It is enriched around spermatid nuclei during postelongation stages and plays an important role in the dynein-dynactin–dependent rostral retention of the nuclei during this period. In addition, DDLC1 colocalizes with dynamin along investment cones and regulates F-actin assembly at this organelle by retaining dynamin along the cones. Interestingly, we found that this process does not require the other subunits of cytoplasmic dynein-dynactin complex. Altogether, these observations suggest that DLC1 could independently regulate multiple cellular functions and established a novel role of this protein in F-actin assembly in Drosophila.

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Bacterial Guanine Nucleotide Exchange Factors SopE-Like and WxxxE Effectors

Infection and Immunity ◽

10.1128/iai.01250-09 ◽

2010 ◽

Vol 78 (4) ◽

pp. 1417-1425 ◽

Cited By ~ 58

Author(s):

Richard Bulgin ◽

Benoit Raymond ◽

James A. Garnett ◽

Gad Frankel ◽

Valerie F. Crepin ◽

...

Keyword(s):

Escherichia Coli ◽

Rho Gtpases ◽

Shigella Flexneri ◽

Small Gtpases ◽

Effector Proteins ◽

Actin Dynamics ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

T3ss Effector

ABSTRACT Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce “the trigger mechanism of cell entry.” Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.

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Selectivity of Guanine Nucleotide Exchange Factor-mediated Cdc42 activation in primary human endothelial cells

10.1101/541219 ◽

2019 ◽

Author(s):

Nathalie R. Reinhard ◽

Sanne van der Niet ◽

Anna Chertkova ◽

Marten Postma ◽

Peter L. Hordijk ◽

...

Keyword(s):

Endothelial Cells ◽

Single Cell ◽

Rho Gtpases ◽

Rho Gtpase ◽

Actin Dynamics ◽

Pleckstrin Homology Domain ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Dh Domain

AbstractThe Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signaling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the full-length GEFs. Our data reveal a specific GEF dependent activation profile, with most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and pRex1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may activate Cdc42, which will be of great value for the field of vascular biology.Abstract FigureGraphical Abstract

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MicroRNA Regulation of the Small Rho GTPase Regulators—Complexities and Opportunities in Targeting Cancer Metastasis

Cancers ◽

10.3390/cancers12051092 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1092 ◽

Cited By ~ 2

Author(s):

Brock A. Humphries ◽

Zhishan Wang ◽

Chengfeng Yang

Keyword(s):

Transcriptional Activation ◽

Rho Gtpases ◽

Cancer Metastasis ◽

Rho Gtpase ◽

Critical Role ◽

Large Family ◽

Actin Dynamics ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange

The small Rho GTPases regulate important cellular processes that affect cancer metastasis, such as cell survival and proliferation, actin dynamics, adhesion, migration, invasion and transcriptional activation. The Rho GTPases function as molecular switches cycling between an active GTP-bound and inactive guanosine diphosphate (GDP)-bound conformation. It is known that Rho GTPase activities are mainly regulated by guanine nucleotide exchange factors (RhoGEFs), GTPase-activating proteins (RhoGAPs), GDP dissociation inhibitors (RhoGDIs) and guanine nucleotide exchange modifiers (GEMs). These Rho GTPase regulators are often dysregulated in cancer; however, the underlying mechanisms are not well understood. MicroRNAs (miRNAs), a large family of small non-coding RNAs that negatively regulate protein-coding gene expression, have been shown to play important roles in cancer metastasis. Recent studies showed that miRNAs are capable of directly targeting RhoGAPs, RhoGEFs, and RhoGDIs, and regulate the activities of Rho GTPases. This not only provides new evidence for the critical role of miRNA dysregulation in cancer metastasis, it also reveals novel mechanisms for Rho GTPase regulation. This review summarizes recent exciting findings showing that miRNAs play important roles in regulating Rho GTPase regulators (RhoGEFs, RhoGAPs, RhoGDIs), thus affecting Rho GTPase activities and cancer metastasis. The potential opportunities and challenges for targeting miRNAs and Rho GTPase regulators in treating cancer metastasis are also discussed. A comprehensive list of the currently validated miRNA-targeting of small Rho GTPase regulators is presented as a reference resource.

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Role of Rho GTPase Interacting Proteins in Subcellular Compartments of Podocytes

International Journal of Molecular Sciences ◽

10.3390/ijms22073656 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3656

Author(s):

Kana Asano-Matsuda ◽

Sajida Ibrahim ◽

Tomoko Takano ◽

Jun Matsuda

Keyword(s):

Rho Gtpases ◽

Rho Gtpase ◽

Small Gtpases ◽

The Body ◽

Actin Dynamics ◽

Future Research ◽

Cellular Functions ◽

Waste Products ◽

Filtration Barrier ◽

Subcellular Compartments

The first step of urine formation is the selective filtration of the plasma into the urinary space at the kidney structure called the glomerulus. The filtration barrier of the glomerulus allows blood cells and large proteins such as albumin to be retained while eliminating the waste products of the body. The filtration barrier consists of three layers: fenestrated endothelial cells, glomerular basement membrane, and podocytes. Podocytes are specialized epithelial cells featured by numerous, actin-based projections called foot processes. Proteins on the foot process membrane are connected to the well-organized intracellular actin network. The Rho family of small GTPases (Rho GTPases) act as intracellular molecular switches. They tightly regulate actin dynamics and subsequent diverse cellular functions such as adhesion, migration, and spreading. Previous studies using podocyte-specific transgenic or knockout animal models have established that Rho GTPases are crucial for the podocyte health and barrier function. However, little attention has been paid regarding subcellular locations where distinct Rho GTPases contribute to specific functions. In the current review, we discuss cellular events involving the prototypical Rho GTPases (RhoA, Rac1, and Cdc42) in podocytes, with particular focus on the subcellular compartments where the signaling events occur. We also provide our synthesized views of the current understanding and propose future research directions.

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Role of Rho-GTPases and their regulatory proteins in glomerular podocyte function

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2013-0135 ◽

2013 ◽

Vol 91 (10) ◽

pp. 773-782 ◽

Cited By ~ 36

Author(s):

Flaviana Mouawad ◽

Harmony Tsui ◽

Tomoko Takano

Keyword(s):

Rho Gtpases ◽

Molecular Mechanisms ◽

Complex Structure ◽

Critical Role ◽

Normal Function ◽

Small Gtpases ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Cellular Functions

Podocytes play a critical role in maintaining glomerular permselectivity. It has been long recognized that their intricate actin-based structures are tightly associated with their normal function; however, the precise mechanisms by which podocytes form and maintain their complex structure had been poorly understood until the intensive investigations on podocyte biology began in 1998, triggered by the breakthrough discovery of nephrin. This review summarizes the recent discoveries of the molecular mechanisms by which the actin cytoskeleton is regulated in podocytes. A particular focus will be on the role of the Rho-family of small GTPases, represented by RhoA, Rac1, and Cdc42. Rho-GTPases are known for their versatile cellular functions, most importantly for the actin regulatory roles. We will also discuss the potential roles of the 3 groups of proteins known to regulate Rho-GTPases, namely GTPase-activating proteins, guanine nucleotide exchange factors, and guanine nucleotide dissociation inhibitors.

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Adenovirus E4orf4 Hijacks Rho GTPase-dependent Actin Dynamics to Kill Cells: A Role for Endosome-associated Actin Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e05-12-1146 ◽

2006 ◽

Vol 17 (7) ◽

pp. 3329-3344 ◽

Cited By ~ 29

Author(s):

Amélie Robert ◽

Nicolas Smadja-Lamère ◽

Marie-Claude Landry ◽

Claudia Champagne ◽

Ryan Petrie ◽

...

Keyword(s):

Rho Gtpases ◽

Actin Polymerization ◽

Rho Gtpase ◽

De Novo ◽

Strong Support ◽

Src Family Kinases ◽

Actin Dynamics ◽

Death Process ◽

Actin Assembly ◽

Orf4 Protein

The adenovirus early region 4 ORF4 protein (E4orf4) triggers a novel death program that bypasses classical apoptotic pathways in human cancer cells. Deregulation of the cell cytoskeleton is a hallmark of E4orf4 killing that relies on Src family kinases and E4orf4 phosphorylation. However, the cytoskeletal targets of E4orf4 and their role in the death process are unknown. Here, we show that E4orf4 translocates to cytoplasmic sites and triggers the assembly of a peculiar juxtanuclear actin–myosin network that drives polarized blebbing and nuclear shrinkage. We found that E4orf4 activates the myosin II motor and triggers de novo actin polymerization in the perinuclear region, promoting endosomes recruitment to the sites of actin assembly. E4orf4-induced actin dynamics requires interaction with Src family kinases and involves a spatial regulation of the Rho GTPases pathways Cdc42/N-Wasp, RhoA/Rho kinase, and Rac1, which make distinct contributions. Remarkably, activation of the Rho GTPases is required for induction of apoptotic-like cell death. Furthermore, inhibition of actin dynamics per se dramatically impairs E4orf4 killing. This work provides strong support for a causal role for endosome-associated actin dynamics in E4orf4 killing and in the regulation of cancer cell fate.

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Wound-induced contractile ring: a model for cytokinesis

Biochemistry and Cell Biology ◽

10.1139/o05-164 ◽

2005 ◽

Vol 83 (6) ◽

pp. 711-720 ◽

Cited By ~ 15

Author(s):

Hassina Darenfed ◽

Craig A Mandato

Keyword(s):

Wound Healing ◽

Rho Gtpases ◽

Wound Closure ◽

Animal Cell ◽

Actin Dynamics ◽

Biological Processes ◽

Animal Cells ◽

Contractile Ring ◽

Small Rho Gtpases ◽

Local Assembly

The actomyosin-based contractile ring is required for several biological processes, such as wound healing and cytokinesis of animal cells. Despite progress in defining the roles of this structure in both wound closure and cell division, we still do not fully understand how an actomyosin ring is spatially and temporally assembled, nor do we understand the molecular mechanism of its contraction. Recent results have demonstrated that microtubule-dependent local assembly of F-actin and myosin-II is present in wound closure and is similar to that in cytokinesis in animal cells. Furthermore, signalling factors such as small Rho GTPases have been shown to be involved in the regulation of actin dynamics during both processes. In this review we address recent findings in an attempt to better understand the dynamics of actomyosin contractile rings during wound healing as compared with the final step of animal cell division.Key words: actomyosin ring, microtubules, cytokinesis, wound healing.

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Actin Cytoskeleton and Regulation of TGFβ Signaling: Exploring Their Links

Biomolecules ◽

10.3390/biom11020336 ◽

2021 ◽

Vol 11 (2) ◽

pp. 336

Author(s):

Roberta Melchionna ◽

Paola Trono ◽

Annalisa Tocci ◽

Paola Nisticò

Keyword(s):

Cancer Progression ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Tissue Homeostasis ◽

Actin Dynamics ◽

Human Tissues ◽

Cellular Functions ◽

Tgfβ Signaling ◽

Physical Mechanisms ◽

And Function

Human tissues, to maintain their architecture and function, respond to injuries by activating intricate biochemical and physical mechanisms that regulates intercellular communication crucial in maintaining tissue homeostasis. Coordination of the communication occurs through the activity of different actin cytoskeletal regulators, physically connected to extracellular matrix through integrins, generating a platform of biochemical and biomechanical signaling that is deregulated in cancer. Among the major pathways, a controller of cellular functions is the cytokine transforming growth factor β (TGFβ), which remains a complex and central signaling network still to be interpreted and explained in cancer progression. Here, we discuss the link between actin dynamics and TGFβ signaling with the aim of exploring their aberrant interaction in cancer.

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Enhanced Production of the Mical Redox Domain for Enzymology and F-actin Disassembly Assays

International Journal of Molecular Sciences ◽

10.3390/ijms22041991 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1991

Author(s):

Jimok Yoon ◽

Heng Wu ◽

Ruei-Jiun Hung ◽

Jonathan R. Terman

Keyword(s):

Nicotinamide Adenine Dinucleotide Phosphate ◽

Actin Dynamics ◽

Specific Substrate ◽

Actin Assembly ◽

Redox Enzymes ◽

Enhanced Production ◽

Reversible Redox ◽

Future Work ◽

Signaling Process

To change their behaviors, cells require actin proteins to assemble together into long polymers/filaments—and so a critical goal is to understand the factors that control this actin filament (F-actin) assembly and stability. We have identified a family of unusual actin regulators, the MICALs, which are flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in Redox reactions. F-actin is a specific substrate for these MICAL Redox enzymes, which oxidize specific amino acids within actin to destabilize actin filaments. Furthermore, this MICAL-catalyzed reaction is reversed by another family of Redox enzymes (SelR/MsrB enzymes)—thereby revealing a reversible Redox signaling process and biochemical mechanism regulating actin dynamics. Interestingly, in addition to the MICALs’ Redox enzymatic portion through which MICALs covalently modify and affect actin, MICALs have multiple other domains. Less is known about the roles of these other MICAL domains. Here we provide approaches for obtaining high levels of recombinant protein for the Redox only portion of Mical and demonstrate its catalytic and F-actin disassembly activity. These results provide a ground state for future work aimed at defining the role of the other domains of Mical — including characterizing their effects on Mical’s Redox enzymatic and F-actin disassembly activity.

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