Polymerization Of Actin Filaments Research Articles

Actin based motility unveiled: How chemical energy is converted into motion

Actin-based motility is a complex process in which the actin-polymerization is the primary force-generating motor machinery. It can produce protrusive forces through actin filaments polymerization and cross-link during lamellipodia protrusion in migrating cells and it is responsible for the intracellular motion of certain pathogens in infected host cells. We propose a multi-physics model for actin-based motility, stemming from continuity equations that account for the actin chemical kinetics. Thermodynamic restrictions are identified, moving from the multiplicative decomposition of the deformation gradient into chemical and elastic parts. Constitutive theory and chemical kinetics are prescribed to finally write governing equations for the multi-physics problem. The field equations are solved numerically with the finite element method. As a proof of concept, a one-dimensional model for actin-based motility of bacteria pathogens is studied.

Dark-induced chloroplast relocation depends on actin filaments in the liverwort Apopellia endiviifolia along with the light- and cold-induced relocations.

Chloroplasts move to the periclinal walls of cells under weak light to harness light energy for photosynthesis and to anticlinal walls to avoid strong light. These responses involve the cytoskeleton components microtubules and/or actin filaments. In the dark, chloroplasts move to the anticlinal cell walls bordering neighbouring cells (dark-positioning response), but this response in various plants normally requires a prolonged dark incubation period, which has hampered analysis. However, we recently demonstrated the dark-positioning response that can be induced after a short period of dark incubation in the liverwort Apopellia endiviifolia. Here, we investigated whether the cytoskeleton components function in the dark-positioning response of A. endiviifolia cells. Microtubules and actin filaments were fluorescentlyvisualised in A. endiviifolia cells and were disrupted following treatment with the microtubule and actin filament polymerisation inhibitors. The dark-positioning response was unaffected in the cells with disrupted microtubules. By contrast, the dark-positioning response was inhibited by the disruption of actin filaments. The disruption of actin filaments also restricted chloroplast mobility during light- and cold-dependent chloroplast movements in A. endiviifolia. Therefore, the dark-positioning response of A. endiviifolia depends solely on an actin filament-associated motility mechanism, as do the light- and cold-dependent chloroplast responses.

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments

Metallothionein 3 (MT-3) is a cysteine-rich metal-binding protein that is expressed in the mammalian central nervous system and kidney. Various reports have posited a role for MT-3 in regulating the actin cytoskeleton by promoting the assembly of actin filaments. We generated purified, recombinant mouse MT-3 of known metal compositions, either with zinc (Zn), lead (Pb), or copper/zinc (Cu/Zn) bound. None of these forms of MT-3 accelerated actin filament polymerization in vitro, either with or without the actin binding protein profilin. Furthermore, using a co-sedimentation assay, we did not observe Zn-bound MT-3 in complex with actin filaments. Cu2+ ions on their own induced rapid actin polymerization, an effect that we attribute to filament fragmentation. This effect of Cu2+ is reversed by adding either EGTA or Zn-bound MT-3, indicating that either molecule can chelate Cu2+ from actin. Altogether, our data indicate that purified recombinant MT-3 does not directly bind actin but it does attenuate the Cu-induced fragmentation of actin filaments.

CLASP2 facilitates dynamic actin filament organization along the microtubule lattice.

Coordination between the microtubule and actin networks is essential for cell motility, neuronal growth cone guidance, and wound healing. Members of the CLASP (cytoplasmic linker-associated protein) family of proteins have been implicated in the cytoskeletal crosstalk between microtubules and actin networks, however, the molecular mechanisms underlying CLASPs role in cytoskeletal coordination are unclear. Here, we investigate CLASP2α's crosslinking function with microtubules and F-actin. Our results demonstrate that CLASP2α crosslinks F-actin to the microtubule lattice in vitro. We find that the crosslinking ability is retained by L-TOG2-S, a minimal construct containing the TOG2 domain and serine-arginine rich region of CLASP2α. Furthermore, CLASP2α promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2α also facilitates dynamic organization of polymerizing actin filaments templated by the microtubule network, with F-actin forming bridges between individual microtubules. Finally, we find that depletion of CLASPs in vascular smooth muscle cells results in disorganized actin fibers and reduced co-alignment of actin fibers with microtubules, suggesting that CLASP and microtubules contribute to higher-order actin structures. Taken together, our results indicate that CLASP2α can directly crosslink F-actin to microtubules, and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.

CLASP2 facilitates dynamic actin filament organization along the microtubule lattice.

Coordination between the microtubule and actin networks is essential for cell motility, neuronal growth cone guidance, and wound healing. Members of the CLASP (cytoplasmic linker-associated protein) family of proteins have been implicated in the cytoskeletal cross-talk between microtubules and actin networks; however, the molecular mechanisms underlying the role of CLASP in cytoskeletal coordination are unclear. Here, we investigate CLASP2α's cross-linking function with microtubules and F-actin. Our results demonstrate that CLASP2α cross-links F-actin to the microtubule lattice in vitro. We find that the cross-linking ability is retained by L-TOG2-S, a minimal construct containing the TOG2 domain and serine-arginine-rich region of CLASP2α. Furthermore, CLASP2α promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2α also facilitates the dynamic organization of polymerizing actin filaments templated by the microtubule network, with F-actin forming bridges between individual microtubules. Finally, we find that depletion of CLASPs in vascular smooth muscle cells results in disorganized actin fibers and reduced coalignment of actin fibers with microtubules, suggesting that CLASP and microtubules contribute to higher-order actin structures. Taken together, our results indicate that CLASP2α can directly cross-link F-actin to microtubules and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.

Mechanism and Prospect of Gastrodin in Osteoporosis, Bone Regeneration, and Osseointegration.

Gastrodin, a traditional Chinese medicine ingredient, is widely used to treat vascular and neurological diseases. However, recently, an increasing number of studies have shown that gastrodin has anti-osteoporosis effects, and its mechanisms of action include its antioxidant effect, anti-inflammatory effect, and anti-apoptotic effect. In addition, gastrodin has many unique advantages in promoting bone healing in tissue engineering, such as inducing high hydrophilicity in the material surface, its anti-inflammatory effect, and pro-vascular regeneration. Therefore, this paper summarized the effects and mechanisms of gastrodin on osteoporosis and bone regeneration in the current research. Here we propose an assumption that the use of gastrodin in the surface loading of oral implants may greatly promote the osseointegration of implants and increase the success rate of implants. In addition, we speculated on the potential mechanisms of gastrodin against osteoporosis, by affecting actin filament polymerization, renin-angiotensin system (RAS) and ferroptosis, and proposed that the potential combination of gastrodin with Mg2+, angiotensin type 2 receptor blockers or artemisinin may greatly inhibit osteoporosis. The purpose of this review is to provide a reference for more in-depth research and application of gastrodin in the treatment of osteoporosis and implant osseointegration in the future.

Cell-Level Analysis Visualizing Photodynamic Therapy with Porphylipoprotein and Talaporphyrin Sodium.

We revealed the difference in the mechanism of photodynamic therapy (PDT) between two photosensitizers: porphylipoprotein (PLP), which has recently attracted attention for its potential to be highly effective in treating cancer, and talaporphyrin sodium (NPe6). (1) NPe6 accumulates in lysosomes, whereas PLP is incorporated into phagosomes formed by PLP injection. (2) PDT causes NPe6 to generate reactive oxygen species, thereby producing actin filaments and stress fibers. In the case of PLP, however, reactive oxygen species generated by PDT remain in the phagosomes until the phagosomal membrane is destroyed, which delays the initiation of RhoA activation and RhoA*/ROCK generation. (4) After the disruption of the phagosomal membrane, however, the outflow of various reactive oxygen species accelerates the production of actin filaments and stress fibers, and blebbing occurs earlier than in the case of NPe6. (5) PLP increases the elastic modulus of cells without RhoA activity in the early stage. This is because phagosomes are involved in polymerizing actin filaments and pseudopodia formation. Considering the high selectivity and uptake of PLP into cancer cells, a larger effect with PDT can be expected by skillfully combining the newly discovered characteristics, such as the appearance of a strong effect at an early stage.

Enhanced sucrose-mediated cryoprotection of siRNA-loaded poly (lactic-co-glycolic acid) nanoparticles

The present study aimed to determine the effects of sucrose on the physical stability, cellular entry pathways and functional efficacy of poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs). PLGA-NPs were synthesized in the absence or presence of 10 % sucrose, using HEI-101, an unmodified small interfering RNA (siRNA), as a drug model. The newly synthesized HEI-101-loaded PLGA-NPs (HEI-101-NPs) were exposed to repeated freeze-thaw cycles and iteratively tested over a six-month evaluation period. The effect of sucrose stabilization on HEI-101-NPs was independently tested in vitro for biocompatibility and cellular uptake in IMO-2B1 cells. Data analyses suggest that, without sucrose, freeze-thaw cycles of HEI-101-NPs resulted in increased particle diameter, increased polydispersity index, and reduced zeta potential. In contrast, a substantial improvement in the physical stability of HEI-101-NPs was observed in the presence of 10 % sucrose. The data revealed that the release of HEI-101 from the PLGA-NPs was governed by polymer erosion and drug diffusion. Data from cellular uptake study in IMO-2B1 cells demonstrated that, 10 % sucrose significantly reduced the inhibitory effect of nocodazole on the microtubule-dependent uptake of PLGA-NPs. In addition, the presence of 10 % sucrose seemed to lessen the inhibitory effect of sodium azide on the energy-dependent uptake of PLGA-NPs. Overall, the current data suggest that the cellular internalization of PLGA-NPs occurred through the polymerization of actin filaments under the control of the microtubules. Our findings reveal cryoprotective effect of 10 % sucrose on HEI-101-NPs that confers marked improvements in the stability, cellular uptake and efficiency for the delivery of biomolecules to inner ear cells.

The conserved Pelado/ZSWIM8 protein regulates actin dynamics by promoting linear actin filament polymerization.

Actin filament polymerization can be branched or linear, which depends on the associated regulatory proteins. Competition for actin monomers occurs between proteins that induce branched or linear actin polymerization. Cell specialization requires the regulation of actin filaments to allow the formation of cell type-specific structures, like cuticular hairs in <i>Drosophila</i>, formed by linear actin filaments. Here, we report the functional analysis of CG34401/<i>pelado</i>, a gene encoding a SWIM domain-containing protein, conserved throughout the animal kingdom, called ZSWIM8 in mammals. Mutant <i>pelado</i> epithelial cells display actin hair elongation defects. This phenotype is reversed by increasing actin monomer levels or by either pushing linear actin polymerization or reducing branched actin polymerization. Similarly, in hemocytes, Pelado is essential to induce filopodia, a linear actin-based structure. We further show that this function of Pelado/ZSWIM8 is conserved in human cells, where Pelado inhibits branched actin polymerization in a cell migration context. In summary, our data indicate that the function of Pelado/ZSWIM8 in regulating actin cytoskeletal dynamics is conserved, favoring linear actin polymerization at the expense of branched filaments.

Flagella-like beating of actin bundles driven by self-organized myosin waves.

Wave-like beating of eukaryotic cilia and flagella-threadlike protrusions found in many cells and microorganisms-is a classic example of spontaneous mechanical oscillations in biology. This type of self-organized active matter raises the question of the coordination mechanism between molecular motor activity and cytoskeletal filament bending. Here we show that in the presence of myosin motors, polymerizing actin filaments self-assemble into polar bundles that exhibit wave-like beating. Importantly, filament beating is associated with myosin density waves initiated at twice the frequency of the actin-bending waves. A theoretical description based on curvature control of motor binding to the filaments and of motor activity explains our observations in a regime of high internal friction. Overall, our results indicate that the binding of myosin to actin depends on the actin bundle shape, providing a feedback mechanism between the myosin activity and filament deformations for the self-organization of large motor filament assemblies.

Biochemical and mechanical regulation of actin dynamics.

Polymerization of actin filaments against membranes produces force for numerous cellular processes, such as migration, morphogenesis, endocytosis, phagocytosis and organelle dynamics. Consequently, aberrant actin cytoskeleton dynamics are linked to various diseases, including cancer, as well as immunological and neurological disorders. Understanding how actin filaments generate forces in cells, how force production is regulated by the interplay between actin-binding proteins and how the actin-regulatory machinery responds to mechanical load are at the heart of many cellular, developmental and pathological processes. During the past few years, our understanding of the mechanisms controlling actin filament assembly and disassembly has evolved substantially. It has also become evident that the activities of key actin-binding proteins are not regulated solely by biochemical signalling pathways, as mechanical regulation is critical for these proteins. Indeed, the architecture and dynamics of the actin cytoskeleton are directly tuned by mechanical load. Here we discuss the general mechanisms by which key actin regulators, often in synergy with each other, control actin filament assembly, disassembly, and monomer recycling. By using an updated view of actin dynamics as a framework, we discuss how the mechanics and geometry of actin networks control actin-binding proteins, and how this translates into force production in endocytosis and mesenchymal cell migration.

Deletion of SM22α disrupts the structure and function of caveolae and T-tubules in cardiomyocytes, contributing to heart failure.

AimsSmooth muscle 22-alpha (SM22α) is an actin-binding protein that plays critical roles in mediating polymerization of actin filaments and stretch sensitivity of cytoskeleton in vascular smooth muscle cells (VSMCs). Multiple lines of evidence indicate the existence of SM22α in cardiomyocytes. Here, we investigated the effect of cardiac SM22α on the membrane architecture and functions of cardiomyocytes to pressure overload.MethodsSM22α knock-out (KO) mice were utilized to assess the role of SM22α in the heart. Echocardiography was used to evaluate cardiac function, transverse aortic constriction (TAC) was used to induce heart failure, cell shortening properties were measured by IonOptix devices in intact cardiomyocytes, Ca2+ sensitivity of myofilaments was measured in permeabilized cardiomyocytes. Confocal microscopy, electron microscopy, western blotting, co-immunoprecipitation (co-IP), Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) techniques were used to perform functional and structural analysis.ResultsSM22α ablation did not alter cardiac function at baseline, but mRNA levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) were increased significantly compared with wild type (WT) controls. The membrane architecture was severely disrupted in SM22α KO cardiomyocytes, with disassembly and flattening of caveolae and disrupted T-tubules. Furthermore, SM22α was co-immunoprecipitated with caveolin-3 (Cav3), and the interaction between Cav3 and actin was significantly reduced in SM22α KO cells. SM22α KO cardiomyocytes displayed asynchronized SR Ca2+ release, significantly increased Ca2+ spark frequency. Additionally, the kinetics of sarcomere shortening was abnormal, accompanied with increased sensitivity and reduced maximum response of myofilaments to Ca2+ in SM22α KO cardiomyocytes. SM22α KO mice were more prone to heart failure after TAC.ConclusionsOur findings identified that SM22α may be required for the architecture and function of caveolae and T-tubules in cardiomyocytes.

Oxidation and reduction of actin: Origin, impact in vitro and functional consequences in vivo

Actin is among the most abundant proteins in eukaryotic cells and assembles into dynamic filamentous networks regulated by many actin binding proteins. The actin cytoskeleton must be finely tuned, both in space and time, to fulfill key cellular functions such as cell division, cell shape changes, phagocytosis and cell migration. While actin oxidation by reactive oxygen species (ROS) at non-physiological levels are known for long to impact on actin polymerization and on the cellular actin cytoskeleton, growing evidence shows that direct and reversible oxidation/reduction of specific actin amino acids plays an important and physiological role in regulating the actin cytoskeleton. In this review, we describe which actin amino acid residues can be selectively oxidized and reduced in many different ways (e.g. disulfide bond formation, glutathionylation, carbonylation, nitration, nitrosylation and other oxidations), the cellular enzymes at the origin of these post-translational modifications, and the impact of actin redox modifications both in vitro and in vivo. We show that the regulated balance of oxidation and reduction of key actin amino acid residues contributes to the control of actin filament polymerization and disassembly at the subcellular scale and highlight how improper redox modifications of actin can lead to pathological conditions.

Global ubiquitinome profiling identifies NEDD4 as a regulator of Profilin 1 and actin remodelling in neural crest cells

The ubiquitin ligase NEDD4 promotes neural crest cell (NCC) survival and stem-cell like properties to regulate craniofacial and peripheral nervous system development. However, how ubiquitination and NEDD4 control NCC development remains unknown. Here we combine quantitative analysis of the proteome, transcriptome and ubiquitinome to identify key developmental signalling pathways that are regulated by NEDD4. We report 276 NEDD4 targets in NCCs and show that loss of NEDD4 leads to a pronounced global reduction in specific ubiquitin lysine linkages. We further show that NEDD4 contributes to the regulation of the NCC actin cytoskeleton by controlling ubiquitination and turnover of Profilin 1 to modulate filamentous actin polymerization. Taken together, our data provide insights into how NEDD4-mediated ubiquitination coordinates key regulatory processes during NCC development.

Cytospin-A Regulates Colorectal Cancer Cell Division and Migration by Modulating Stability of Microtubules and Actin Filaments.

Simple SummaryIn this study, we report the effects of depleting cytospin-A (CYTSA), also known as the sperm antigen with calponin homology and coiled-coil domain (SPECC1L) protein, on the proliferation and migration of colorectal cancer (CRC) cells. Mutations in this protein have been previously linked to different developmental disorders. In our studies, depletion of CYTSA in various CRC cells led to significant decreases in proliferation, increases in cell death, and increased formation of multinucleated cells. Knocking down CYTSA also led to severe inhibition of CRC cell migration and invasion. These effects could be related to a significant decrease in the stability of microtubules and alterations in polymerized actin filaments in CYTSA depleted CRC cells. Our studies, for the first time, provide evidence suggesting that targeting CYTSA may be a novel therapeutic strategy for patients with CRC.Proteins that interact with cytoskeletal elements play important roles in cell division and are potentially important targets for therapy in cancer. Cytospin-A (CYTSA), a protein known to interact with actin and microtubules, has been previously described to be important in various developmental disorders, including oblique facial clefting. We hypothesized that CYTSA plays an important role in colorectal cancer (CRC) cell division. The effects of CYTSA depletion on CRC cell proliferation were analyzed using cell growth assays, microscopic analyses of live and fixed cells, and time-lapse imaging. CYTSA depletion led to inhibition of cell proliferation, significant increases in CRC cell death, and accumulation of doublet cells during and following cell division. Depletion of CYTSA also resulted in strong inhibition of CRC cell migration and invasion. Mechanistically, CYTSA depletion resulted in significant decreases in the stability of microtubules and altered polymerization of actin filaments in CRC cells. Finally, bioinformatic analyses were performed to determine the correlation between CYTSA expression and survival of patients with CRC. Interestingly, a strong correlation between high CYTSA expression and poor survival was observed in the TCGA adenocarcinoma data set but not in an independent data set. Since inhibiting CYTSA significantly reduces CRC cell proliferation, migration, and invasion, targeting CYTSA may be a potential novel therapeutic option for patients with metastatic CRC.

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-poly-L-proline (PLP) interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.

Lymphocyte Polarization During Immune Synapse Assembly: Centrosomal Actin Joins the Game.

Interactions among immune cells are essential for the development of adaptive immune responses. The immunological synapse (IS) provides a specialized platform for integration of signals and intercellular communication between T lymphocytes and antigen presenting cells (APCs). In the T cell the reorganization of surface molecules at the synaptic interface is initiated by T cell receptor binding to a cognate peptide-major histocompatibility complex on the APC surface and is accompanied by a polarized remodelling of the cytoskeleton and centrosome reorientation to a subsynaptic position. Although there is a general agreement on polarizing signals and mechanisms driving centrosome reorientation during IS assembly, the primary events that prepare for centrosome repositioning remain largely unexplored. It has been recently shown that in resting lymphocytes a local polymerization of filamentous actin (F-actin) at the centrosome contributes to anchoring this organelle to the nucleus. During early stages of IS formation centrosomal F-actin undergoes depletion, allowing for centrosome detachment from the nucleus and its polarization towards the synaptic membrane. We recently demonstrated that in CD4+ T cells the reduction in centrosomal F-actin relies on the activity of a centrosome-associated proteasome and implicated the ciliopathy-related Bardet-Biedl syndrome 1 protein in the dynein-dependent recruitment of the proteasome 19S regulatory subunit to the centrosome. In this short review we will feature our recent findings that collectively provide a new function for BBS proteins and the proteasome in actin dynamics, centrosome polarization and T cell activation.

Actin Alpha 2 Downregulation Inhibits Neural Stem Cell Proliferation and Differentiation into Neurons through Canonical Wnt/β-Catenin Signaling Pathway.

Our previous study has shown that actin alpha 2 (ACTA2) is expressed in NSC and ACTA2 downregulation inhibits NSC migration by increasing RhoA expression and decreasing the expression of Rac1 to curb actin filament polymerization. Given that proliferation and differentiation are the two main characteristics of NSC, the role of ACTA2 downregulation in the proliferation and differentiation of NSC remains elusive. Here, the results demonstrated that ACTA2 downregulation using ACTA2 siRNA held the potential of inhibiting NSC proliferation using cell counting kit-8 (CCK8) and immunostaining. Then, our data illustrated that ACTA2 downregulation attenuated NSC differentiation into neurons, while directing NSC into astrocytes and oligodendrocytes using immunostaining and immunoblotting. Thereafter, the results revealed that the canonical Wnt/β-catenin pathway was involved in the effect of ACTA2 downregulation on the proliferation and differentiation of NSC through upregulating p-β-catenin and decreasing β-catenin due to inactivating GSK-3β, while this effect could be partially abolished with administration of CHIR99012, a GSK-3 inhibitor. Collectively, these results indicate that ACTA2 downregulation inhibits NSC proliferation and differentiation into neurons through inactivation of the canonical Wnt/β-catenin pathway. The aim of the present study is to elucidate the role of ACTA2 in proliferation and differentiation of NSC and to provide an intervention target for promoting NSC proliferation and properly directing NSC differentiation.

Design principles of endocytic actin architecture and force production

Actin filament polymerization produces force to shape cellular membranes. In clathrin-mediated endocytosis (CME), a branched actin network assembles and disassembles over ∼20 s to help internalize the clathrin-coated plasma membrane. Such a branched network can self-assemble into a polarized array to internalize the membrane (Akamatsu et al., 2020). However, the relationship between the architecture of the branched actin cytoskeleton, its evolution over time, and its adaptation to changing loads remains poorly understood.

Protective mitochondrial fission induced by stress-responsive protein GJA1-20k.

The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.