The dynein light chain protein Tda2 functions as a dimerization engine to regulate actin capping protein during endocytosis

Clathrin- and actin-mediated endocytosis is a fundamental process in eukaryotic cells. Previously, we discovered Tda2 as a new yeast dynein light chain that works with Aim21 to regulate actin assembly during endocytosis. Here, we show Tda2 functions as a dimerization engine bringing two Aim21 molecules together using a novel binding surface different than the canonical dynein light chain ligand binding groove. Point mutations on either protein that diminish the Tda2-Aim21 interaction in vitro cause the same in vivo phenotype as TDA2 deletion showing reduced actin capping protein recruitment and increased filamentous actin at endocytic sites. Remarkably, chemically induced dimerization of Aim21 rescues the endocytic phenotype of TDA2 deletion. We also uncovered a capping protein interacting motif in Aim21, expanding its function to a fundamental cellular pathway and showing such motif exists outside mammalian cells. Furthermore, specific disruption of this motif causes the same deficit of actin capping protein recruitment and increased filamentous actin at endocytic sites as AIM21 deletion. Thus, the data indicates the Tda2-Aim21 complex functions in actin assembly primarily through capping protein regulation. Collectively, our results provide a mechanistic view of the Tda2-Aim21 complex and its function in actin network regulation at endocytic sites.

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Novel function of a dynein light chain in actin assembly during clathrin-mediated endocytosis

The Journal of Cell Biology ◽

10.1083/jcb.201604123 ◽

2017 ◽

Vol 216 (8) ◽

pp. 2565-2580 ◽

Cited By ~ 6

Author(s):

Kristen B. Farrell ◽

Seth McDonald ◽

Andrew K. Lamb ◽

Colette Worcester ◽

Olve B. Peersen ◽

...

Keyword(s):

Light Chain ◽

Protein Family ◽

Dependent Manner ◽

Dynein Light Chain ◽

Filamentous Actin ◽

Capping Protein ◽

Dynein Motor ◽

Actin Capping Protein ◽

Endocytic Machinery ◽

Polyproline Motif

Clathrin- and actin-mediated endocytosis is essential in eukaryotic cells. In this study, we demonstrate that Tda2 is a novel protein of the endocytic machinery necessary for normal internalization of native cargo in yeast. Tda2 has not been classified in any protein family. Unexpectedly, solving the crystal structure of Tda2 revealed it belongs to the dynein light chain family. However, Tda2 works independently of the dynein motor complex and microtubules. Tda2 forms a tight complex with the polyproline motif–rich protein Aim21, which interacts physically with the SH3 domain of the Arp2/3 complex regulator Bbc1. The Tda2–Aim21 complex localizes to endocytic sites in a Bbc1- and filamentous actin–dependent manner. Importantly, the Tda2–Aim21 complex interacts directly with and facilitates the recruitment of actin-capping protein, revealing barbed-end filament capping at endocytic sites to be a regulated event. Thus, we have uncovered a new layer of regulation of the actin cytoskeleton by a member of a conserved protein family that has not been previously associated with a function in endocytosis.

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HIPK4 is essential for murine spermiogenesis

eLife ◽

10.7554/elife.50209 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 4

Author(s):

J Aaron Crapster ◽

Paul G Rack ◽

Zane J Hellmann ◽

Austen D Le ◽

Christopher M Adams ◽

...

Keyword(s):

Cellular Transformation ◽

Filamentous Actin ◽

Interacting Protein ◽

Cultured Fibroblasts ◽

Vitro Fertilization ◽

Capping Protein ◽

Actin Capping Protein ◽

Abnormal Head ◽

Actin Capping

Mammalian spermiogenesis is a remarkable cellular transformation, during which round spermatids elongate into chromatin-condensed spermatozoa. The signaling pathways that coordinate this process are not well understood, and we demonstrate here that homeodomain-interacting protein kinase 4 (HIPK4) is essential for spermiogenesis and male fertility in mice. HIPK4 is predominantly expressed in round and early elongating spermatids, and Hipk4 knockout males are sterile, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Hipk4 mutant sperm have reduced oocyte binding and are incompetent for in vitro fertilization, but they can still produce viable offspring via intracytoplasmic sperm injection. Optical and electron microscopy of HIPK4-null male germ cells reveals defects in the filamentous actin (F-actin)-scaffolded acroplaxome during spermatid elongation and abnormal head morphologies in mature spermatozoa. We further observe that HIPK4 overexpression induces branched F-actin structures in cultured fibroblasts and that HIPK4 deficiency alters the subcellular distribution of an F-actin capping protein in the testis, supporting a role for this kinase in cytoskeleton remodeling. Our findings establish HIPK4 as an essential regulator of sperm head shaping and potential target for male contraception.

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The expression of human testis‐specific actin capping protein predicts in vitro fertilization outcomes: A novel biomarker of sperm function for assisted reproductive technology

Reproductive Medicine and Biology ◽

10.1002/rmb2.12407 ◽

2021 ◽

Author(s):

Yusuke Inagaki ◽

Shinichiro Fukuhara ◽

Sohei Kuribayashi ◽

Koichi Okada ◽

Yosuke Sekii ◽

...

Keyword(s):

In Vitro Fertilization ◽

Assisted Reproductive Technology ◽

Reproductive Technology ◽

Human Testis ◽

Sperm Function ◽

Vitro Fertilization ◽

Capping Protein ◽

Actin Capping Protein ◽

Actin Capping

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Gelsolin-like actin-capping protein has prognostic value and promotes tumorigenesis and epithelial-mesenchymal transition via the Hippo signaling pathway in human bladder cancer

Therapeutic Advances in Medical Oncology ◽

10.1177/1758835919841235 ◽

2019 ◽

Vol 11 ◽

pp. 175883591984123 ◽

Cited By ~ 5

Author(s):

Lyu Zhaojie ◽

Liu Yuchen ◽

Chen Miao ◽

Chen Yacun ◽

Wu Shayi ◽

...

Keyword(s):

Bladder Cancer ◽

Epithelial Mesenchymal Transition ◽

Histologic Subtype ◽

Mesenchymal Transition ◽

Capping Protein ◽

Incidence And Mortality ◽

Actin Capping Protein ◽

Actin Capping

Background: Transitional cell carcinoma (TCC) of the bladder, the major histologic subtype of bladder cancer, is increasing in incidence and mortality, which requires the identification of effective biomarkers. Actin-regulating proteins have recently been proposed as important antitumor druggable targets. As a gelsolin-family actin-modulating protein, CAPG (gelsolin-like actin-capping protein) generated great interest due to its crucial effects in various biological and physiological processes; however, the role and mechanism of CAPG in TCCs remain unknown. Materials and methods: Bioinformatic analysis and immunohistochemistry of clinical specimens were performed to detect the expression level of CAPG. Both in vitro and in vivo assays were used to determine the oncogenic effect of CAPG in TCCs. Male 4–5-week-old BALB/c nude mice were used for in vivo tumorigenesis assays, while SCID mice were used for in vivo metastatic assays. Affymetrix microarray was used to identify the underlying molecular mechanism. Western blot and immunofluorescence were used to validate the expression and localization of proteins. Results: CAPG was frequently upregulated in TCCs and associated with clinical aggressiveness and worse prognosis. Functional assays demonstrated that CAPG could contribute to the tumorigenesis, metastasis and epithelial-mesenchymal transition (EMT) of TCCs both in vitro and in vivo. A novel mechanism that CAPG promoted TCC development via inactivating the Hippo pathway, leading to a nucleus translocation of Yes-associated protein was suggested. Conclusions: The current study identified CAPG as a novel and critical oncogene in TCCs, supporting the pursuit of CAPG as a potential target for TCC intervention.

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Cap Z(36/32), a barbed end actin-capping protein, is a component of the Z-line of skeletal muscle.

The Journal of Cell Biology ◽

10.1083/jcb.105.1.371 ◽

1987 ◽

Vol 105 (1) ◽

pp. 371-379 ◽

Cited By ~ 91

Author(s):

J F Casella ◽

S W Craig ◽

D J Maack ◽

A E Brown

Keyword(s):

Electron Microscopy ◽

Skeletal Muscle ◽

Actin Filaments ◽

Biological Activities ◽

Capping Protein ◽

Actin Capping Protein ◽

In Vitro Effects ◽

Actin Capping

Various biological activities have been attributed to actin-capping proteins based on their in vitro effects on actin filaments. However, there is little direct evidence for their in vivo activities. In this paper, we show that Cap Z(36/32), a barbed end, actin-capping protein isolated from muscle (Casella, J. F., D. J. Maack, and S. Lin, 1986, J. Biol. Chem., 261:10915-10921) is localized to the barbed ends of actin filaments by electron microscopy and to the Z-line of chicken skeletal muscle by indirect immunofluorescence and electron microscopy. Since actin filaments associate with the Z-line at their barbed ends, these findings suggest that Cap Z(36/32) may play a role in regulating length, orienting, or attaching actin filaments to Z-discs.

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Vertebrate Isoforms of Actin Capping Protein β Have Distinct Functions in Vivo

The Journal of Cell Biology ◽

10.1083/jcb.147.6.1287 ◽

1999 ◽

Vol 147 (6) ◽

pp. 1287-1298 ◽

Cited By ~ 53

Author(s):

Marilyn C. Hart ◽

John A. Cooper

Keyword(s):

Actin Filaments ◽

Cell Periphery ◽

Actin Assembly ◽

Severe Phenotype ◽

Intercalated Disc ◽

Capping Protein ◽

Intercalated Discs ◽

Actin Capping Protein ◽

Actin Capping

Actin capping protein (CP) binds barbed ends of actin filaments to regulate actin assembly. CP is an α/β heterodimer. Vertebrates have conserved isoforms of each subunit. Muscle cells contain two β isoforms. β1 is at the Z-line; β2 is at the intercalated disc and cell periphery in general. To investigate the functions of the isoforms, we replaced one isoform with another using expression in hearts of transgenic mice. Mice expressing β2 had a severe phenotype with juvenile lethality. Myofibril architecture was severely disrupted. The β2 did not localize to the Z-line. Therefore, β1 has a distinct function that includes interactions at the Z-line. Mice expressing β1 showed altered morphology of the intercalated disc, without the lethality or myofibril disruption of the β2-expressing mice. The in vivo function of CP is presumed to involve binding barbed ends of actin filaments. To test this hypothesis, we expressed a β1 mutant that poorly binds actin. These mice showed both myofibril disruption and intercalated disc remodeling, as predicted. Therefore, CPβ1 and CPβ2 each have a distinct function that cannot be provided by the other isoform. CPβ1 attaches actin filaments to the Z-line, and CPβ2 organizes the actin at the intercalated discs.

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Physiological role of the interaction between CARMIL1 and capping protein

Molecular Biology of the Cell ◽

10.1091/mbc.e13-05-0270 ◽

2013 ◽

Vol 24 (19) ◽

pp. 3047-3055 ◽

Cited By ~ 20

Author(s):

Marc Edwards ◽

Yun Liang ◽

Taekyung Kim ◽

John A. Cooper

Keyword(s):

Cell Migration ◽

Mammalian Cells ◽

Physiological Role ◽

Physiological Significance ◽

Actin Assembly ◽

Capping Protein ◽

And Control ◽

In Cells

The regulation of free barbed ends is central to the control of dynamic actin assembly and actin-based motility in cells. Capping protein (CP) is known to regulate barbed ends and control actin assembly in cells. The CARMIL family of proteins can bind and inhibit CP in vitro, but the physiological significance of the interaction of CARMIL with CP in cells is poorly understood. Mammalian cells lacking CARMIL1 have defects in lamellipodia, macropinocytosis, cell migration, and Rac1 activation. Here we investigate the physiological significance of the CARMIL1–CP interaction, using a point mutant with a well-defined biochemical defect. We find that the CARMIL1–CP interaction is essential for the assembly of lamellipodia, the formation of ruffles, and the process of macropinocytosis. In contrast, the interaction of CARMIL1 with CP shows little to no importance for other functions of CARMIL1, including localization of CARMIL1 to the membrane, activation of Rac1, and cell migration. One implication is that lamellipodia are only marginally important for cell migration in a wound-healing model. The results also suggest that the ability of CARMIL1 to inhibit CP in cells may be regulated.

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Comparative analysis of CPI-motif regulation of biochemical functions of actin capping protein

10.1101/2020.02.06.936211 ◽

2020 ◽

Author(s):

Patrick McConnell ◽

Marlene Mekel ◽

Alexander G. Kozlov ◽

Olivia L. Mooren ◽

Timothy M. Lohman ◽

...

Keyword(s):

Comparative Analysis ◽

Amino Acid Residues ◽

Actin Assembly ◽

Protein Families ◽

Capping Protein ◽

Functional Assays ◽

Biochemical Assays ◽

Actin Capping Protein ◽

End Capping ◽

Actin Capping

ABSTRACTThe heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families to a) bind to CP, b) allosterically inhibit barbed-end capping by CP, and c) allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino-acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs(1, 2) across all protein families (1, 2). These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and actin assembly near membranes.

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CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish

10.1101/2020.09.27.315655 ◽

2020 ◽

Author(s):

Benjamin C. Stark ◽

Yuanyuan Gao ◽

Lakyn Belk ◽

Matthew A. Culver ◽

Bo Hu ◽

...

Keyword(s):

Cell Migration ◽

Cluster Formation ◽

Actin Assembly ◽

Capping Protein ◽

Capping Proteins ◽

Myosin I ◽

Actin Capping Protein ◽

Endodermal Cells ◽

Actin Capping

AbstractCell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants show impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, affecting the morphogenesis of Kupffer’s vesicle (KV). Mutant KVs are smaller and display decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This first in vivo animal study of CARMIL3 reveals its new role for CARMIL3 during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.

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