Actin-associated Proteins Research Articles

Synaptopodin: a key regulator of Hebbian plasticity

Synaptopodin, an actin-associated protein found in a subset of dendritic spines in telencephalic neurons, has been described to influence both functional and morphological plasticity under various plasticity paradigms. Synaptopodin is necessary and sufficient for the formation of the spine apparatus, stacks of smooth endoplasmic reticulum cisternae. The spine apparatus is a calcium store that locally regulates calcium dynamics in response to different patterns of activity and is also thought to be a site for local protein synthesis. Synaptopodin is present in ~30% of telencephalic large dendritic spines in vivo and in vitro highlighting the heterogeneous microanatomy and molecular architecture of dendritic spines, an important but not well understood aspect of neuroplasticity. In recent years, it has become increasingly clear that synaptopodin is a formidable regulator of multiple mechanisms essential for learning and memory. In fact, synaptopodin appears to be the decisive factor that determines whether plasticity can occur, acting as a key regulator for synaptic changes. In this review, we summarize the current understanding of synaptopodin’s role in various forms of Hebbian synaptic plasticity.

Candidate proteins interacting with cytoskeleton in cells from the basal airway epithelium in vitro.

Introduction: The cytoskeleton consists of actin, microtubules, septins, and intermediate filaments and, in most cells, is anchored to an extracellular matrix. Each cell has a unique arrangement of this network and readjusts it from time to time. To investigate the regulation of these reorganizations, we identified interactors from extracts of four cultured lines representing basal cells from the airway epithelium. Methods: After immunoprecipitation with an antibody against keratin 17, samples were processed by liquid chromatography and tandem mass spectrometry. Samples not undergoing antibody-mediated capture were processed in parallel. Results: The main keratins of basal cells, namely, Krt14 (type I) and Krt5 (type II), constituted 67% of the total keratin recovered. Several other intermediate filament proteins, nestin, lamin-B1, and prelamin A/C, were present but not enriched upon immunoprecipitation. Although the class of armadillo-repeat proteins was represented by beta-catenin1 and plakoglobin, other desmosome plaque constituents were absent. Large cytolinkers were represented by the spectraplakin, microtubule-actin cross-linking factor (Macf1), which was enriched by immunoprecipitation, and the plakin, plectin, which was not enriched. Subunits of actin filaments and microtubules, along with numerous proteins associated with them, were recovered in both immunoprecipitated samples and those lacking the capture step. Coefficients of determination were computed based on abundance. The actin-associated proteins, alpha-spectrin and brain-specific angiogenesis inhibitor (Baiaip2l), were modestly correlated with keratin abundance but highly correlated with one another and with the keratin-binding protein, annexin A2. This interaction network resembled the pedestal formed by pathogenic Escherichia coli. Microtubule-associated proteins, dynamin 1-like protein and cytoplasmic dynein 1 heavy chain (Dync1h1), were enriched by immunoprecipitation, suggesting association with keratins, whereas kinesin-1 heavy chain and microtubule-associated protein retinitis pigmentosa 1 (EB1), were not enriched. Dync1h1 abundance was negatively correlated with that of all the septins, suggesting resemblance to a known antagonistic septin-dynein 1 relationship on microtubules. Conclusion: The cell lines showed remarkable uniformity with respect to the candidates interacting with cytoskeleton. The alpha-spectrin-Baiap2l network may link actin filaments to keratin precursor particles. A smaller interaction network centered on Dync1h1 was negatively correlated with all spectrin-Baiap2l constituents, suggesting that it and its binding partners are excluded from the pedestal-like domain.

KLHL17 differentially controls the expression of AMPA- and KA-type glutamate receptors to regulate dendritic spine enlargement.

Kelch-like family member 17 (KLHL17), an actin-associated adaptor protein, is linked to neurological disorders, including infantile spasms and autism spectrum disorders. The key morphological feature of Klhl17-deficient neurons is impaired dendritic spine enlargement, resulting in the amplitude of calcium events being increased. Our previous studies have indicated an involvement of F-actin and the spine apparatus in KLHL17-mediated dendritic spine enlargement. Here, we show that KLHL17 further employs different mechanisms to control the expression of two types of glutamate receptors, that is, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainate receptors (KARs), to regulate dendritic spine enlargement and calcium influx. We deployed proteomics to reveal that KLHL17 interacts with N-ethylmaleimide-sensitive fusion protein (NSF) in neurons, with this interaction of KLHL17 and NSF enhancing NSF protein levels. Consistent with the function of NSF in regulating the surface expression of AMPAR, Klhl17 deficiency limits the surface expression of AMPAR, but not its total protein levels. The NSF pathway also contributes to synaptic F-actin distribution and the dendritic spine enlargement mediated by KLHL17. KLHL17 is known to act as an adaptor mediating degradation of the KAR subunit GluK2 by the CUL3 ubiquitin ligase complex, and Klhl17 deficiency impairs activity-dependent degradation of GluK2. Herein, we further demonstrate that GluK2 is critical to the increased amplitude of calcium influx in Klhl17-deficient neurons. Moreover, GluK2 is also involved in KLHL17-regulated dendritic spine enlargement. Thus, our study reveals that KLHL17 controls AMPAR and KAR expression via at least two mechanisms, consequently regulating dendritic spine enlargement. The regulatory effects of KLHL17 on these two glutamate receptors likely contribute to neuronal features in patients suffering from certain neurological disorders.

The role of PALLD-STAT3 interaction in megakaryocyte differentiation and thrombocytopenia treatment.

Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a crucial transcription factor in regulating megakaryocyte differentiation, yet the precise mechanism of its activation remains unclear. PALLD, an actin-associated protein, has been increasingly recognized for its essential functions in multiple biological processes. This study revealed that megakaryocyte/plateletspecific knockout of PALLD in mice exhibited thrombocytopenia due to diminished platelet biogenesis. In megakaryocytes, PALLD deficiency led to impaired proplatelet formation and polyploidization, ultimately weakening their differentiation for platelet production. Mechanistic studies demonstrated that PALLD bound to STAT3 and interacted with its DNA-binding domain (DBD) and Src homology 2 (SH2) domain via Immunoglobulin domain 3 (Ig3). Moreover, the absence of PALLD attenuated STAT3 Y705 phosphorylation and impeded STAT3 nuclear translocation. Based on the PALLD-STAT3 binding sequence, we designed a peptide C-P3, which can facilitate megakaryocyte differentiation and accelerate platelet production in vivo. In conclusion, this study highlights the pivotal role of PALLD in megakaryocyte differentiation and proposes a novel approach for treating thrombocytopenia by targeting the PALLD-STAT3 interaction.

#2695 A Polycystin-1-Bicaudal C1 complex regulates focal adhesion function through integrin beta-1, F-actin organisation and local translation in HKE cells

Abstract Background and Aims Variants in Bicc1, result in a recessive murine PKD phenotype (bpk, jcpk) by uncertain mechanisms. In a recent study, we reported genetic and biochemical evidence of novel interactions between PKD1, PKD2 and BICC1 in Xenopus, mouse and man suggesting that BICC1 is a modifier of the ADPKD phenotype (submitted). In this study, our major aim was to investigate the molecular mechanisms underlying cystogenesis that result from loss of the normal interaction between the PKD1 protein, Polycystin-1 (PC1), a cell surface receptor and Bicaudal C1 (BICC1), an RNA-binding protein. Methods Isogenic PKD1 and BICC1 CRISP-Cas9 KO cells were generated from a conditionally-immortalised wild-type human proximal tubular line (UCL93); RNA-seq was performed in PKD1 and BICC1 KO cells to identify commonly altered genes and pathways; MS pull-down was conducted in HEK293 cells with BICC1-HA to identify BICC1 interacting proteins; cellular phenotypes in normal and KO cells were assessed through adhesion and migration assays, alongside Traction Force Microscopy (TFM); proximal ligation assays (PLA) to confirm protein-protein interactions; single-molecule FISH (smFISH) to localize mRNA transcripts; puromycin labeling with a proximity ligation assay (Puro-PLA) to detect sites of translation of specific mRNAs. Results Consistent with a functional interaction, loss of PC1 resulted in lower BICC1 levels and vice-versa in single KO (SKO) cells. In migrating cells, BICC1 was expressed at the leading edge where it co-localised with β1-integrin (ITGB1) by PLA, suggesting enrichment at focal adhesion sites (FA). BICC1 KO cells had no cilia phenotype but instead displayed striking disorganization of stress fibres and aster-like structures, features which could be completely rescued by lentiviral Bicc1-HA re-expression. Gene set enrichment and pathway analysis confirmed that F-actin organization and actin-associated proteins (AAP) were commonly dysregulated in SKO cells. BICC1 was also found to interact ITGB1, several components of an integrin-associated complex and proteins involved in actin turnover by MS pull-down. Both SKO cells showed major abnormalities of cell attachment, detachment and directional migration indicative of functional alterations in cell-matrix and cell-cell adhesion. We confirmed that FA structure and function were altered by reduced co-localisation of several AAPs (including TNS1 and FBLIM1) and by TFM. BICC1 directly interacted with F-actin and FBLIM1 in pull-down assays and with FBLIM1 by RNA-IP. Finally, we found evidence of FBLIM1 mRNA by smFISH and of local FBLIM1 translation (Puro-PLA) at FA sites in wild-type cells, both of which were markedly reduced in SKO cells. Conclusion Our data reveal the key roles for actin dynamics and local translation as novel mechanisms regulated by PC1 and BICC1 in the pathogenesis of ADPKD. We demonstrate that BICC1 and PC1 are co-regulated, forming a PC1-BICC1 complex critical for maintaining the structural integrity and function of FAs in normal kidney epithelial cells. Loss of this interaction results in major alterations in cell adhesion, migration and stress fibre organisation, features typical of the ADPKD cellular phenotype. BICC1 plays a dual role in this process via protein-protein interactions with ITGB1, F-actin and other AAPs, and through protein-RNA interactions regulating the local translation of key AAPs (FBLIM1) at FA sites.

Myosin-1C augments endothelial secretion of von Willebrand factor by linking contractile actomyosin machinery to the plasma membrane

AbstractBlood endothelial cells control the hemostatic and inflammatory response by secreting von Willebrand factor (VWF) and P-selectin from storage organelles called Weibel-Palade bodies (WPBs). Actin-associated motor proteins regulate this secretory pathway at multiple points. Before fusion, myosin Va forms a complex that anchors WPBs to peripheral actin structures, allowing for the maturation of content. After fusion, an actomyosin ring/coat is recruited and compresses the WPB to forcibly expel the largest VWF multimers. Here, we provide, to our knowledge, the first evidence for the involvement of class I myosins during regulated VWF secretion. We show that the unconventional myosin-1C (Myo1c) is recruited after fusion via its pleckstrin homology domain in an actin-independent process. This provides a link between the actin ring and phosphatidylinositol 4,5-bisphosphate (PIP2) at the membrane of the fused organelle and is necessary to ensure maximal VWF secretion. This is an active process requiring Myo1c ATPase activity because inhibition of class I myosins using the inhibitor pentachloropseudilin or expression of an ATPase-deficient Myo1c rigor mutant perturbs the expulsion of VWF and alters the kinetics of the exocytic actin ring. These data offer a novel insight into the control of an essential physiological process and provide a new way in which it can be regulated.

Synaptopodin is required for long-term depression at Schaffer collateral-CA1 synapses

Synaptopodin (SP), an actin-associated protein found in telencephalic neurons, affects activity-dependant synaptic plasticity and dynamic changes of dendritic spines. While being required for long-term depression (LTD) mediated by metabotropic glutamate receptor (mGluR-LTD), little is known about its role in other forms of LTD induced by low frequency stimulation (LFS-LTD) or spike-timing dependent plasticity (STDP). Using electrophysiology in ex vivo hippocampal slices from SP-deficient mice (SPKO), we show that absence of SP is associated with a deficit of LTD at Sc-CA1 synapses induced by LFS-LTD and STDP. As LTD is known to require AMPA- receptors internalization and IP3-receptors calcium signaling, we tested by western blotting and immunochemistry if there were changes in their expression which we found to be reduced. While we were not able to induce LTD, long-term potentiation (LTP), albeit diminished in SPKO, can be recovered by using a stronger stimulation protocol. In SPKO we found no differences in NMDAR, which are the primary site of calcium signalling to induce LTP. Our study shows, for the first time, the key role of the requirement of SP to allow induction of activity-dependant LTD at Sc-CA1 synapses.

Benefits and challenges of reconstituting the actin cortex.

The cell's ability to change shape is a central feature in many cellular processes, including cytokinesis, motility, migration, and tissue formation. The cell constructs a network of contractile proteins underneath the cell membrane to form the cortex, and the reorganization of these components directly contributes to cellular shape changes. The desire to mimic these cell shape changes to aid in the creation of a synthetic cell has been increasing. Therefore, membrane-based reconstitution experiments have flourished, furthering our understanding of the minimal components the cell uses throughout these processes. Although biochemical approaches increased our understanding of actin, myosin II, and actin-associated proteins, using membrane-based reconstituted systems has further expanded our understanding of actin structures and functions because membrane-cortex interactions can be analyzed. In this review, we highlight the recent developments in membrane-based reconstitution techniques. We examine the current findings on the minimal components needed to recapitulate distinct actin structures and functions and how they relate to the cortex's impact on cellular mechanical properties. We also explore how co-processing of computational models with wet-lab experiments enhances our understanding of these properties. Finally, we emphasize the benefits and challenges inherent to membrane-based, reconstitution assays, ranging from the advantage of precise control over the system to the difficulty of integrating these findings into the complex cellular environment.

Loss of synaptopodin impairs mGluR5 and protein synthesis-dependent mGluR-LTD at CA3-CA1 synapses.

Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) is an important form of synaptic plasticity that occurs in many regions of the central nervous system and is the underlying mechanism for several learning paradigms. In the hippocampus, mGluR-LTD is manifested by the weakening of synaptic transmission and elimination of dendritic spines. Interestingly, not all spines respond or undergo plasticity equally in response to mGluR-LTD. A subset of dendritic spines containing synaptopodin (SP), an actin-associated protein is critical for mGluR-LTD and protects spines from elimination through mGluR1 activity. The precise cellular function of SP is still enigmatic and it is still unclear how SP contributes to the functional aspect of mGluR-LTD despite its modulation of the structural plasticity. In this study, we show that the lack of SP impairs mGluR-LTD by negatively affecting the mGluR5-dependent activity. Such impairment of mGluR5 activity is accompanied by a significant decrease of surface mGluR5 level in SP knockout (SPKO) mice. Intriguingly, the remaining mGluR-LTD becomes a protein synthesis-independent process in the SPKO and is mediated instead by endocannabinoid signaling. These data indicate that the postsynaptic protein SP can regulate the locus of expression of mGluR-LTD and provide insight into our understanding of spine/synapse-specific plasticity.

The Cell Permeant Phosphopetpidemimetic of VASP Alleviates Motor Function Deficits After Experimental Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) due to the rupture of an intracranial aneurysm leads to delayed vasospasm and neuroischemia, which can result in profound neurologic deficit and death. Therapeutic options after SAH are currently limited to hemodynamic optimization and nimodipine, which have limited clinical efficacy. Experimental SAH results in cerebral vasospasm have demonstrated the downregulation of nitric oxide (NO)-protein kinase G (PKG) signaling elements. VP3 is a novel cell permeant phosphopeptide mimetic of VASP, a substrate of PKG and an actin-associated protein that modulates vasorelaxation in vascular smooth muscle cells. In this study, we determined that intravenous administration of high doses of VP3 did not induce systemic hypotension in rats except at the maximal soluble dose, implying that VP3 is well-tolerated and has a wide therapeutic window. Using a single cisterna magna injection rat model of SAH, we demonstrated that intravenous administration of low-dose VP3 after SAH improved neurologic deficits for up to 14days as determined by the rotarod test. These findings suggest that strategies aimed at targeting the cerebral vasculature with VP3 may improve neurologic deficits associated with SAH.

Myosin VI powers self-organization of branched contractile actin network

The actomyosin cytoskeletal network is responsible for a variety of fundamental cellular processes. Assembly and maintenance of actin networks involve an array of associated regulatory proteins for polymerization, branching, crosslinking and contractility-driven self-organization. In this study, we make the unexpected discovery in vitro that myosin VI and myosin X, motor proteins specialized in vesicle transport and filopodia formation, are capable of crosslinking and self-organizing actin into higher-order contractile structures in the absence of other actin-associated proteins. Moreover, myosin VI alone can initiate actin elongation and branching, and assemble branched force-generating networks from crosslinked actin polymers. Additional architectural control is provided by the actin crosslinking proteins α-actinin and fascin. Our data identify critical stages of tension-mediated connectivity in network development and provide a model system for further exploration of the nonequilibrium mechanics of actomyosin self-organization.

Dynamic actin/septin network in megakaryocytes coordinates proplatelet elaboration.

Megakaryocytes (MK) undergo extensive cytoskeletal rearrangements as they give rise to platelets. While cortical microtubule sliding has been implicated in proplatelet formation, the role of the actin cytoskeleton in proplatelet elongation is less understood. It is assumed that actin filament reorganization is important for platelet generation given that mouse models with mutations in actin-associated proteins exhibit thrombocytopenia. However, due to the essential role of the actin network during MK development, a differential understanding of the contribution of the actin cytoskeleton on proplatelet release is lacking. Here, we reveal that inhibition of actin polymerization impairs the formation of elaborate proplatelets by hampering proplatelet extension and bead formation along the proplatelet shaft, which was mostly independent of changes in cortical microtubule sliding. We identify Cdc42 and its downstream effectors, septins, as critical regulators of intracellular actin dynamics in MK, inhibition of which, similarly to inhibition of actin polymerization, impairs proplatelet movement and beading. Super-resolution microscopy revealed a differential association of distinctive septins with the actin and microtubule cytoskeleton, respectively, which was disrupted upon septin inhibition and diminished intracellular filamentous actin dynamics. In vivo, septins, similarly to F-actin, were subject to changes in expression upon enforcing proplatelet formation through prior platelet depletion. In summary, we demonstrate that a Cdc42/septin axis is not only important for MK maturation and polarization, but is further required for intracellular actin dynamics during proplatelet formation.

14-3-3 Proteins stabilize actin and vimentin filaments to maintain processes in renal glomerular podocyte.

14-3-3 proteins are a ubiquitously expressed family of adaptor proteins. Despite exhibiting high sequence homology, several 14-3-3 isoforms have isoform-specific binding partners and roles. We reported that 14-3-3β interacts with FKBP12 and synaptopodin to maintain the structure of actin fibers in podocytes. However, the precise localization and differential role of 14-3-3 isoforms in kidneys are unclear. Herein, we showed that 14-3-3β in glomeruli was restricted in podocytes, and 14-3-3σ in glomeruli was expressed in podocytes and mesangial cells. Although 14-3-3β was dominantly co-localized with FKBP12 in the foot processes, a part of 14-3-3β was co-localized with Par3 at the slit diaphragm. 14-3-3β interacted with Par3, and FKBP12 bound to 14-3-3β competitively with Par3. Deletion of 14-3-3β enhanced the interaction of Par3 with Par6 in podocytes. Gene silencing for 14-3-3β altered the structure of actin fibers and process formation. 14-3-3β and synaptopodin expression was decreased in podocyte injury models. In contrast, 14-3-3σ in podocytes was expressed in the primary processes. 14-3-3σ interacted with vimentin but not with the actin-associated proteins FKBP12 and synaptopodin. Gene silencing for 14-3-3σ altered the structure of vimentin fibers and process formation. 14-3-3σ and vimentin expression was increased in the early phase of podocyte injury models but was decreased in the late stage. Together, the localization of 14-3-3β at actin cytoskeleton plays a role in maintaining the foot processes and the Par complex in podocytes. In contrast, 14-3-3σ at vimentin cytoskeleton is essential for maintaining primary processes.

Single particle tracking reveals SARS-CoV-2 regulating and utilizing dynamic filopodia for viral invasion

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry mechanism has been explored, little is known about how SARS-CoV-2 regulates the subcellular structural remodeling to invade multiple organs and cell types. Here, we unveil how SARS-CoV-2 boosts and utilizes filopodia to enter the target cells by real-time imaging. Using SARS-CoV-2 single virus-like particle (VLP) tracking in live cells and sparse deconvolution algorithm, we uncover that VLPs utilize filopodia to reach the entry site in two patterns, “surfing” and “grabbing”, which avoid the virus from randomly searching on the plasma membrane. Moreover, combining mechanical simulation, we elucidate that the formation of virus-induced filopodia and the retraction speed of filopodia depend on cytoskeleton dynamics and friction resistance at the substrate surface caused by loading-virus gravity, respectively. Further, we discover that the entry process of SARS-CoV-2 via filopodia depends on Cdc42 activity and actin-associated proteins fascin, formin, and Arp2/3. Together, our results highlight that the spatial–temporal regulation of actin cytoskeleton by SARS-CoV-2 infection makes filopodia as a highway for virus entry and potentiates it as an antiviral target.

Interaction of Filamin C With Actin Is Essential for Cardiac Development and Function.

FLNC (filamin C), a member of the filamin family predominantly expressed in striated muscles, plays a crucial role in bridging the cytoskeleton and ECM (extracellular matrix) in cardiomyocytes, thereby maintaining heart integrity and function. Although genetic variants within the N-terminal ABD (actin-binding domain) of FLNC have been identified in patients with cardiomyopathy, the precise contribution of the actin-binding capability to FLNC's function in mammalian hearts remains poorly understood. We conducted in silico analysis of the 3-dimensional structure of mouse FLNC to identify key amino acid residues within the ABD that are essential for FLNC's actin-binding capacity. Subsequently, we performed coimmunoprecipitation and immunofluorescent assays to validate the in silico findings and assess the impact of these mutations on the interactions with other binding partners and the subcellular localization of FLNC. Additionally, we generated and analyzed knock-in mouse models in which the FLNC-actin interaction was completely disrupted by these mutations. Our findings revealed that F93A/L98E mutations completely disrupted FLNC-actin interaction while preserving FLNC's ability to interact with other binding partners ITGB1 (β1 integrin) and γ-SAG (γ-sarcoglycan), as well as maintaining FLNC subcellular localization. Loss of FLNC-actin interaction in embryonic cardiomyocytes resulted in embryonic lethality and cardiac developmental defects, including ventricular wall malformation and reduced cardiomyocyte proliferation. Moreover, disruption of FLNC-actin interaction in adult cardiomyocytes led to severe dilated cardiomyopathy, enhanced lethality and dysregulation of key cytoskeleton components. Our data strongly support the crucial role of FLNC as a bridge between actin filaments and ECM through its interactions with actin, ITGB1, γ-SAG, and other associated proteins in cardiomyocytes. Disruption of FLN-actin interaction may result in detachment of actin filaments from the extracellular matrix, ultimately impairing normal cardiac development and function. These findings also provide insights into mechanisms underlying cardiomyopathy associated with genetic variants in FLNC ABD and other regions.

Clinical and Molecular Aspects of C2orf71/PCARE in Retinal Diseases.

Mutations in the photoreceptor-specific C2orf71 gene (also known as photoreceptor cilium actin regulator protein PCARE) cause autosomal recessive retinitis pigmentosa type 54 and cone-rod dystrophy. No treatments are available for patients with C2orf71 retinal ciliopathies exhibiting a severe clinical phenotype. Our understanding of the disease process and the role of PCARE in the healthy retina significantly limits our capacity to transfer recent technical developments into viable therapy choices. This study summarizes the current understanding of C2orf71-related retinal diseases, including their clinical manifestations and an unclear genotype-phenotype correlation. It discusses molecular and functional studies on the photoreceptor-specific ciliary PCARE, focusing on the photoreceptor cell and its ciliary axoneme. It is proposed that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane during the development of a new outer segment disk in photoreceptor cells. This review also introduces various cellular and animal models used to model these diseases and provides an overview of potential treatments.

Chiral growth of adherent filopodia

Adherent filopodia are elongated finger-like membrane protrusions, extending from the edges of diverse cell types and participating in cell adhesion, spreading, migration, and environmental sensing. The formation and elongation of filopodia are driven by the polymerization of parallel actin filaments, comprising the filopodia cytoskeletal core. Here, we report that adherent filopodia, formed during the spreading of cultured cells on galectin-8-coated substrates, tend to change the direction of their extension in a chiral fashion, acquiring a left-bent shape. Cryoelectron tomography examination indicated that turning of the filopodia tip to the left is accompanied by the displacement of the actin core bundle to the right of the filopodia midline. Reduction of the adhesion to galectin-8 by treatment with thiodigalactoside abolished this filopodia chirality. By modulating the expression of a variety of actin-associated filopodia proteins, we identified myosin-X and formin DAAM1 as major filopodia chirality promoting factors. Formin mDia1, actin filament elongation factor VASP, and actin filament cross-linker fascin were also shown to be involved. Thus, the simple actin cytoskeleton of filopodia, together with a small number of associated proteins are sufficient to drive a complex navigation process, manifested by the development of left-right asymmetry in these cellular protrusions.

A census of actin-associated proteins in humans.

Actin filaments help in maintaining the cell structure and coordinating cellular movements and cargo transport within the cell. Actin participates in the interaction with several proteins and also with itself to form the helical filamentous actin (F-actin). Actin-binding proteins (ABPs) and actin-associated proteins (AAPs) coordinate the actin filament assembly and processing, regulate the flux between globular G-actin and F-actin in the cell, and help maintain the cellular structure and integrity. We have used protein-protein interaction data available through multiple sources (STRING, BioGRID, mentha, and a few others), functional annotation, and classical actin-binding domains to identify actin-binding and actin-associated proteins in the human proteome. Here, we report 2482 AAPs and present an analysis of their structural and sequential domains, functions, evolutionary conservation, cellular localization, abundance, and tissue-specific expression patterns. This analysis provides a base for the characterization of proteins involved in actin dynamics and turnover in the cell.

Abstract 331: Mitochondrial transfer from astrocytes drives glioblastoma tumorigenicity

Abstract Mitochondrial transfer in the central nervous system occurs from astrocytes to neurons in stroke. Mitochondrial exchange has also been reported among tumor cells in glioblastoma (GBM), the most common primary brain tumor. However, the role of mitochondrial transfer from non-neoplastic cells in the surrounding microenvironment to GBM remains poorly understood. We hypothesized that mitochondrial transfer from these non-neoplastic to GBM cells supports tumor metabolism and growth. Using transgenic mice expressing fluorophore-tagged mitochondria, we found that ~50% of orthotopically-implanted mouse GBM cells acquire host mitochondria. Brain-resident cells, mainly astrocytes, but not infiltrating immune cells were the primary mitochondrial donors in vivo and in vitro. Mitochondrial transfer also occurred from immortalized human astrocytes to a broad array of patient-derived xenograft (PDX) models of GBM in vitro at rates of 15-35%. GBM cells that acquired mitochondria expressed higher levels of the ATP-synthase subunit ATP5A and produced more ATP, while metabolomics revealed upregulated amino acid metabolism in recipient cells. In vivo, mouse GBM cells that acquired mitochondria were more likely to be in G2/M proliferative cell cycle phases. We observed a similar effect in PDX that acquired astrocyte mitochondria from co-cultures in vitro. To mechanistically link increased proliferation specifically to mitochondrial transfer, we isolated astrocyte mitochondria by differential centrifugation and found that addition and uptake of cell-free mitochondria in human GBM cells recapitulated the increased proliferation. Using sorted mouse and human GBM cells with/without astrocyte mitochondrial acquisition, we further found that mitochondrial transfer promoted in vitro self-renewal and in vivo tumorigenicity, leading to significant reduction in survival and increased penetrance in orthotopic GBM models. Transfer in mouse and human systems was contact-dependent and was abrogated by physical separation of donor and recipient cells by transwell inserts. We visualized contact-dependent transfer across actin-based intercellular connections consistent with previously reported microtubes. We confirmed the critical role of actin and the actin-associated protein, growth-associated protein 43 (GAP43) in facilitating mitochondrial transfer by showing that pharmacologic inhibition and genetic knockdown (respectively) significantly decreased the rate of mitochondrial transfer. Taken together, mitochondrial transfer comprises a fundamental, protumorigenic mechanism of GBM, enhancing metabolic activity and driving tumor cell proliferation. Further elucidating the molecular machinery regulating astrocyte mitochondrial transfer and its downstream protumorigenic effects will lead to therapeutic opportunities targeting this understudied tumor microenvironment interaction. Citation Format: Dionysios C. Watson, Defne Bayik, Simon Storevik, Shannon S. Moreino, Samuel S. Sprowls, Gauravi Deshpande, Palavalasa Sravya, Costas A. Lyssiotis, Daniel R. Wahl, Hrvoje Miletic, Justin D. Lathia. Mitochondrial transfer from astrocytes drives glioblastoma tumorigenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 331.

Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities.

Palladin (PALLD) belongs to the PALLD/myopalladin (MYPN)/myotilin family of actin-associated immunoglobulin-containing proteins in the sarcomeric Z-line. PALLD is ubiquitously expressed in several isoforms, and its longest 200 kDa isoform, predominantly expressed in striated muscle, shows high structural homology to MYPN. MYPN gene mutations are associated with human cardiomyopathies, whereas the role of PALLD in the heart has remained unknown, partly due to embryonic lethality of PALLD knockout mice. In a yeast two-hybrid screening, CARP/Ankrd1 and FHOD1 were identified as novel interaction partners of PALLD's N-terminal region. To study the role of PALLD in the heart, we generated conditional (cPKO) and inducible (cPKOi) cardiomyocyte-specific PALLD knockout mice. While cPKO mice exhibited no pathological phenotype, ablation of PALLD in adult cPKOi mice caused progressive cardiac dilation and systolic dysfunction, associated with reduced cardiomyocyte contractility, intercalated disc abnormalities, and fibrosis, demonstrating that PALLD is essential for normal cardiac function. Double cPKO and MYPN knockout (MKO) mice exhibited a similar phenotype as MKO mice, suggesting that MYPN does not compensate for the loss of PALLD in cPKO mice. Altered transcript levels of MYPN and PALLD isoforms were found in myocardial tissue from human dilated and ischemic cardiomyopathy patients, whereas their protein expression levels were unaltered.