GJA1-20k, an internally translated isoform of Connexin 43, is an actin capping protein

Previously, we identified that GJA1-20k, an internally translated isoform of Connexin 43, mediates an actin-dependent protective form of mitochondrial fission (Shimura, Nuebel et al. 2021). We found that when GJA1-20k is present, bands of actin surround mitochondria at locations enriched with GJA1-20k, inducing mitochondrial fission which generates less oxygen free radicals, protecting hearts subjected to ischemia-reperfusion injury. Here, we report that GJA1-20k is a direct actin binding protein and thereby identify the mechanism by which GJA1-20k is able to recruit and stabilize actin filaments around the mitochondria. Surprisingly, GJA1-20k functions as a canonical actin capping protein, producing both truncated actin puncta and stabilized actin filaments. GJA1-20k contains an RPEL-like actin binding motif, and we confirm with both computational modeling and biochemistry, that this domain is crucial for actin capping. The actin capping functionality of GJA1-20k adds GJA1-20k to the family of proteins that regulate actin dynamics. As a stress responsive protein, GJA1-20k can help explain cytoskeletal dependent responses to cellular stress, from delivery of channels to affecting mitochondrial size and function.

Functional Requirements for a Samd14-Capping Protein Complex in Stress Erythropoiesis

Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes. Paracrine signals – involving cooperation between SCF/c-Kit signaling and other signaling inputs – are required for the increased erythroid precursor activity in anemia. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene increases the regenerative capacity of the erythroid system and promotes stress-dependent c-Kit signaling. However, the mechanism underlying Samd14’s role in stress erythropoiesis is unknown. We identified a protein-protein interaction between Samd14 and the α- and β heterodimers of the F-actin capping protein (CP) complex. Knockdown of the CP β subunit increased erythroid maturation in ex vivo cultures and decreased colony forming potential of stress erythroid precursors. In a genetic complementation assay for Samd14 activity, our results revealed that the Samd14-CP interaction is a determinant of erythroid precursor cell levels and function. Samd14-CP promotes SCF/c-kit signaling in CD71med spleen erythroid precursors. Given the roles of c-Kit signaling in hematopoiesis and Samd14 in c-Kit pathway activation, this mechanism may have pathological implications in acute/chronic anemia.

Actin capping protein regulates actomyosin contractility to maintain germline architecture in C. elegans

Actin dynamics play an important role in the morphogenesis of cells and tissues, yet the control of actin filament growth takes place at the molecular level. A challenge in the field is to link the molecular function of actin regulators with their physiological function. Here, we report the in vivo role of the actin capping protein CAP-1 in the C. elegans germline. We show that CAP-1 is associated with actomyosin structures in the cortex and rachis, where it keeps the level of contractility in check. A 60% reduction in the level of CAP-1 leads to a 2-fold increase in F-actin and non-muscle myosin II and only a 30% increase in Arp2/3. CAP-1 depletion leads to severe structural defects in the syncytial germline and oocytes, which can be rescued by reducing myosin activity. Thus, we uncover a physiological role for actin capping protein in maintaining C. elegans fertility by regulating the level of actomyosin contractility.

Structure and Function of an Atypical Homodimeric Actin Capping Protein from the Malaria Parasite

Apicomplexan parasites, such as Plasmodium spp., rely on an unusual actomyosin motor, termed glideosome, for motility and host cell invasion. The actin filaments are maintained by a small set of essential regulators, which provide control over actin dynamics in the different stages of the parasite life cycle. Actin filament capping proteins (CPs) are indispensable heterodimeric regulators of actin dynamics. CPs have been extensively characterized in higher eukaryotes, but their role and functional mechanism in Apicomplexa remain enigmatic. Here, we present the first crystal structure of a homodimeric CP from the malaria parasite and compare the homo- and heterodimeric CP structures in detail. Despite retaining several characteristics of a canonical CP, the homodimeric Plasmodium berghei (Pb)CP exhibits crucial differences to the canonical heterodimers. Both homo- and heterodimeric PbCPs regulate actin dynamics in an atypical manner, facilitating rapid turnover of parasite actin, without affecting its critical concentration. Homo- and heterodimeric PbCPs show partially redundant activities, possibly to rescue actin filament capping in life cycle stages where the β-subunit is downregulated,. Our data suggest that the homodimeric PbCP also influences actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a β-subunit, as the asymmetric PbCP homodimer lacks structural elements essential for canonical barbed end interactions suggesting a novel CP binding mode. These findings will facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium.

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.

E3 Ubiquitin Ligase UBR5 Promotes the Metastasis of Pancreatic Cancer via Destabilizing F-Actin Capping Protein CAPZA1

The ubiquitin-proteasome system (UPS) is a regulated mechanism of intracellular protein degradation and turnover, and its dysfunction is associated with various diseases including cancer. UBR5, an E3 ubiquitin ligase, is emerging as an important regulator of the UPS in cancers, but its role in pancreatic cancer is poorly understood. Here, we show that UBR5 is significantly upregulated in pancreatic cancer tissues. High UBR5 expression is correlated with increased lymph node metastasis and poor survival of patients. The loss-of-function and gain-of-function studies demonstrated that UBR5 substantially enhanced the in vitro migratory and invasive ability of pancreatic cancer cells. UBR5 knockdown also markedly inhibited in vivo cancer metastasis in the liver metastatic model of pancreatic cancer in nude mice, suggesting UBR5 as a potent metastatic promoter in pancreatic cancer. Furthermore, using co-immunoprecipitation combined with mass spectrometry analyses, CAPZA1, a member of F-actin capping protein α subunit family, was identified as a novel substrate of UBR5. UBR5 overexpression could promote the degradation of CAPZA1 via the UPS and induce the accumulation of F-actin, which has been described as an essential molecular event during the process of CAPZA1 deficiency-induced cancer cells migration and invasion. UBR5 knockdown significantly increased the intracellular level of CAPZA1 and CAPZA1 downregulation largely reversed the UBR5 knockdown-induced suppression of cell migration and invasion in pancreatic cancer cells. Collectively, our findings unveil UBR5 as a novel and critical regulator of pancreatic cancer metastasis and highlight the potential for UBR5-CAPZA1 axis as a therapeutic target for preventing metastasis in pancreatic cancer patients, especially in those with increased UBR5 expression.

The structure of the actin filament uncapping complex mediated by twinfilin

Uncapping of actin filaments is essential for driving polymerization and depolymerization dynamics from capping protein–associated filaments; however, the mechanisms of uncapping leading to rapid disassembly are unknown. Here, we elucidated the x-ray crystal structure of the actin/twinfilin/capping protein complex to address the mechanisms of twinfilin uncapping of actin filaments. The twinfilin/capping protein complex binds to two G-actin subunits in an orientation that resembles the actin filament barbed end. This suggests an unanticipated mechanism by which twinfilin disrupts the stable capping of actin filaments by inducing a G-actin conformation in the two terminal actin subunits. Furthermore, twinfilin disorders critical actin-capping protein interactions, which will assist in the dissociation of capping protein, and may promote filament uncapping through a second mechanism involving V-1 competition for an actin-binding surface on capping protein. The extensive interactions with capping protein indicate that the evolutionary conserved role of twinfilin is to uncap actin filaments.