Crystal Structures of the Full-Length Murine and Human Gasdermin D Reveal Mechanisms of Autoinhibition, Lipid Binding, and Oligomerization.

Gasdermin D (GSDMD) is an effector molecule for pyroptosis downstream of canonical and noncanonical inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases triggers the oligomerization and lipid binding by its N-terminal domain, which assembles membrane pores, whereas its C-terminal domain binds the N-terminal domain to inhibit pyroptosis. Despite recent progress in our understanding of the structure and function of the murine gasdermin A3 (mGSDMA3), the molecular mechanisms of GSDMD activation and regulation remain poorly characterized. Here, we report the crystal structures of the full-length murine and human GSDMDs, which reveal the architecture of the GSDMD N-terminal domains and demonstrate distinct and common features of autoinhibition among gasdermin family members utilizing their ß1-ß2 loops. Disruption of the intramolecular domain interface enhanced pyroptosis, whereas mutations at the predicted lipid-binding or oligomerization surface reduced cytolysis. Our study provides a framework for understanding the autoinhibition, lipid binding, and oligomerization of GSDMD by using overlapping interfaces.

Disease-associated mutations in Drp1 have fundamentally different effects on the mitochondrial fission machinery.

Patient mutations have been identified throughout dynamin-related protein 1 (Drp1), the key protein mediator of mitochondrial fission. These changes generally impact young children and often result in severe neurological defects and, in some instances, death. Until now, the underlying functional defect leading to patient phenotypes has been largely speculative. We therefore analyzed six disease-associated mutations throughout the GTPase and middle domains (MD) of Drp1. The MD plays a role in Drp1 oligomerization, and three mutations in this region were predictably impaired in self-assembly. However, another mutant in this region (F370C) retained oligomerization capability on pre-curved membranes despite being assembly-limited in solution. Instead, this mutation impaired membrane remodeling of liposomes, which highlights the importance of Drp1 in generating local membrane curvature before fission. Two GTPase domain mutations were also observed in different patients. The G32A mutation was impaired in GTP hydrolysis both in solution and in the presence of lipid but remains capable of self-assembly on these lipid templates. The G223V mutation also exhibited decreased GTPase activity and was able to assemble on pre-curved lipid templates; however, this change impaired membrane remodeling of unilamellar liposomes similar to F370C. This demonstrates that the Drp1 GTPase domain also contributes to self-assembly interactions that drive membrane curvature. Overall, the functional defects caused by mutations in Drp1 are highly variable even for mutations that reside within the same functional domain. This study provides a framework for characterizing additional Drp1 mutations to provide a comprehensive understanding of functional sites within this essential protein.

Conserved Pib2 regions have distinct roles in TORC1 regulation at the vacuole.

TORC1 is a critical controller of cell growth in eukaryotes. In yeast (Saccharomyces cerevisiae), the presence of nutrients is signaled to TORC1 by several upstream regulatory sensors that together coordinate TORC1 activity. TORC1 localizes to both vacuolar and endosomal membranes, where differential signaling occurs. This localization is mimicked by Pib2, a key upstream TORC1 regulator that is essential for TORC1 reactivation after nutrient starvation or pharmacological inhibition. Pib2 has both positive and negative effects on TORC1 activity, but the mechanisms remain poorly understood. Here, we pinpoint the Pib2 inhibitory function on TORC1 to residues within short, conserved N-terminal regions. We also show that the Pib2 C-terminal regions, helical region E and tail, are essential for TORC1 reactivation. Furthermore, the Pib2 FYVE domain plays a role in vacuolar localization, but it is surprisingly unnecessary for recovery from rapamycin exposure. Using chimeric Pib2 targeting constructs, we show that endosomal localization is not necessary for TORC1 reactivation and cell growth after rapamycin treatment. Thus, a comprehensive molecular dissection of Pib2 demonstrates that each of its conserved regions differentially contribute to Pib2-mediated regulation of TORC1 activity.

Coincident Phosphatidic Acid Interaction Restrains Drp1 in Mitochondrial Division.

Mitochondria divide to control their size, distribution, turnover, and function. Dynamin-related protein 1 (Drp1) is a critical mechanochemical GTPase that drives constriction during mitochondrial division. It is generally believed that mitochondrial division is regulated during recruitment of Drp1 to mitochondria and its oligomerization into a division apparatus. Here, we report an unforeseen mechanism that regulates mitochondrial division by coincident interactions of Drp1 with the head group and acyl chains of phospholipids. Drp1 recognizes the head group of phosphatidic acid (PA) and two saturated acyl chains of another phospholipid by penetrating into the hydrophobic core of the membrane. The dual phospholipid interactions restrain Drp1 via inhibition of oligomerization-stimulated GTP hydrolysis that promotes membrane constriction. Moreover, a PA-producing phospholipase, MitoPLD, binds Drp1, creating a PA-rich microenvironment in the vicinity of a division apparatus. Thus, PA controls the activation of Drp1 after the formation of the division apparatus.

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission.

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission.

Pten associates with important gene regulatory network to fine-tune Müller glia-mediated zebrafish retina regeneration.

Unlike mammals, zebrafish possess a remarkable ability to regenerate damaged retina after an acute injury. Retina regeneration in zebrafish involves the induction of Müller glia-derived progenitor cells (MGPCs) exhibiting stem cell-like characteristics, which are capable of restoring all retinal cell-types. The induction of MGPC through Müller glia-reprograming involves several cellular, genetic and biochemical events soon after a retinal injury. Despite the knowledge on the importance of Phosphatase and tensin homolog (Pten), which is a dual-specificity phosphatase and tumor suppressor in the maintaining of cellular homeostasis, its importance during retina regeneration remains unknown. Here, we explored the importance of Pten during zebrafish retina regeneration. The Pten gets downregulated upon retinal injury and is absent from the MGPCs, which is essential to trigger Akt-mediated cellular proliferation essential for retina regeneration. We found that the downregulation of Pten in the post-injury retina accelerates MGPCs formation, while its overexpression restricts the regenerative response. We observed that Pten regulates the proliferation of MGPCs not only through Akt pathway but also by Mmp9/Notch signaling. Mmp9-activity is essential to induce the proliferation of MGPCs in the absence of Pten. Lastly, we show that expression of Pten is fine-tuned through Mycb/histone deacetylase1 and Tgf-ß signaling. The present study emphasizes on the stringent regulation of Pten and its crucial involvement during the zebrafish retina regeneration.

Molecular characterization of linker and loop-mediated structural modulation and hinge motion in the C4-C5 domains of cMyBPC.

INTRODUCTION: The central C4 and C5 domains (C4C5) of cardiac myosin binding protein C (cMyBPC) contain a flexible interdomain linker and a cardiac-isoform specific loop. However, their importance in the functional regulation of cMyBPC has not been extensively studied. METHODS AND RESULTS: We expressed recombinant C4C5 proteins with deleted linker and loop regions and performed biophysical experiments to determine each of their structural and dynamic roles. We show that the linker and C5 loop regions modulate the secondary structure and thermal stability of C4C5. Furthermore, we provide evidence through extended molecular dynamics simulations and principle component analyses that C4C5 can adopt a completely bent or latched conformation. The simulation trajectory and interaction network analyses reveal that the completely bent conformation of C4C5 exhibits a specific pattern of residue-level interactions. Therefore, we propose a "hinge-and-latch" mechanism where the linker allows a great degree of flexibility and bending, while the loop aids in achieving a completely bent and latched conformation. Although this may be one of many bent positions that C4C5 can adopt, we illustrate for the first time in molecular detail that this type of large scale conformational change can occur in the central domains of cMyBPC. CONCLUSIONS: Our hinge-and-latch mechanism demonstrates that the linker and loop regions participate in dynamic modulation of cMyBPC's motion and global conformation. These structural and dynamic features may contribute to muscle isoform-specific regulation of actomyosin activity, and have potential implications regarding its ability to propagate or retract cMyBPC's regulatory N-terminal domains.

Association of symptom characteristics and comorbid conditions with viral RNA positivity of Covid-19 patients in Kasaragod district in Kerala, India: A retrospective cohort study.

Background Symptoms of Covid-19 are known to be non-specific ranging from asymptomatic cases to severe illness affecting multiple organ systems. The duration of viral RNA positivity and transmission varies in individuals. We describe the association between symptom characteristics and comorbid conditions with viral RNA positivity of SARSCoV-2 affected individuals. Methods We conducted a record-based retrospective cohort study of 179 patients found to be positive for Covid-19 in Kasaragod district in Kerala. We included details of all patients found positive during the initial phases of the pandemic and recorded details regarding symptoms, duration of viral RNA positivity and the occurrence of transmission. The data were analysed using SPSS. Results Any symptom was present in 68%. Fever (43%) was the most common symptom while 50% had at least one respiratory symptom. Increased duration of viral RNA positivity was found to be associated with presence of comorbid conditions. The majority of individuals who transmitted disease (75%) had some symptom, predominantly a respiratory symptom. Conclusion Respiratory symptoms are seen in half of the patients and viral RNA positivity was for a longer duration in patients with comorbid conditions.

Mitochondrial dynamics: The dynamin superfamily and execution by collusion.

Distinct dynamin superfamily GTPases catalyze the constant fission and fusion of the elaborate mitochondrial networks that navigate the eukaryotic cytoplasm. Long believed to be the singular handiwork of dynamin-related protein 1 (Drp1), a cytosolic family member that transiently localizes to the mitochondrial surface, the execution of mitochondrial fission is now arguably believed to entail membrane remodeling events that are initiated upstream of Drp1 by ER-associated cytoskeletal networks and completed downstream by the prototypical dynamin, dynamin 2 (Dyn2). Recent developments in the field have also placed a sharp focus on the membrane microenvironment around the division apparatus and the potential facilitatory role of specific lipids in mitochondrial fission. Here, I will review current progress, as well as highlight the most visible gaps in knowledge, in elucidating the varied functions of the dynamin superfamily in the coordinated events of mitochondrial fission and fusion. The essential roles of protein and lipid cofactors are also highlighted.

The glutathione degrading enzyme, Chac1, is required for calcium signaling in developing zebrafish: redox as an upstream activator of calcium.

Calcium signaling is essential for embryonic development but the signals upstream of calcium are only partially understood. Here, we investigate the role of the intracellular glutathione redox potential in calcium signaling using the Chac1 protein of zebrafish. A member of the γ-glutamylcyclotransferase family of enzymes, the zebrafish Chac1 is a glutathione-degrading enzyme that acts only on reduced glutathione. The zebrafish chac1 expression was seen early in development, and in the latter stages, in the developing muscles, brain and heart. The chac1 knockdown was embryonic lethal, and the developmental defects were seen primarily in the myotome, brain and heart where chac1 was maximally expressed. The phenotypes could be rescued by the WT Chac1 but not by the catalytically inactive Chac1 that was incapable of degrading glutathione. The ability of chac1 to alter the intracellular glutathione redox potential in the live animals was examined using Grx1-roGFP2. The chac1 morphants lacked the increased degree of cellular oxidation seen in the WT zebrafish. As calcium is also known to be critical for the developing myotomes, brain and heart, we further investigated if the chac1 knockdown phenotypes were a consequence of the lack of calcium signals. We observed using GCaMP6s, that calcium transients normally seen in the developing embryos were strongly attenuated in these knockdowns. The study thus identifies Chac1 and the consequent change in intracellular glutathione redox potential as important upstream activators of calcium signaling during development.

Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission.

Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.

Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor.

Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction.

Distinct Splice Variants of Dynamin-related Protein 1 Differentially Utilize Mitochondrial Fission Factor as an Effector of Cooperative GTPase Activity.

Multiple isoforms of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) arise from the alternative splicing of its single gene-encoded pre-mRNA transcript. Among these, the longer Drp1 isoforms, expressed selectively in neurons, bear unique polypeptide sequences within their GTPase and variable domains, known as the A-insert and the B-insert, respectively. Their functions remain unresolved. A comparison of the various biochemical and biophysical properties of the neuronally expressed isoforms with that of the ubiquitously expressed, and shortest, Drp1 isoform (Drp1-short) has revealed the effect of these inserts on Drp1 function. Utilizing various biochemical, biophysical, and cellular approaches, we find that the A- and B-inserts distinctly alter the oligomerization propensity of Drp1 in solution as well as the preferred curvature of helical Drp1 self-assembly on membranes. Consequently, these sequences also suppress Drp1 cooperative GTPase activity. Mitochondrial fission factor (Mff), a tail-anchored membrane protein of the mitochondrial outer membrane that recruits Drp1 to sites of ensuing fission, differentially stimulates the disparate Drp1 isoforms and alleviates the autoinhibitory effect imposed by these sequences on Drp1 function. Moreover, the differential stimulatory effects of Mff on Drp1 isoforms are dependent on the mitochondrial lipid, cardiolipin (CL). Although Mff stimulation of the intrinsically cooperative Drp1-short isoform is relatively modest, CL-independent, and even counter-productive at high CL concentrations, Mff stimulation of the much less cooperative longest Drp1 isoform (Drp1-long) is robust and occurs synergistically with increasing CL content. Thus, membrane-anchored Mff differentially regulates various Drp1 isoforms by functioning as an allosteric effector of cooperative GTPase activity.

The mechanoenzymatic core of dynamin-related protein 1 comprises the minimal machinery required for membrane constriction.

Mitochondria are dynamic organelles that continually undergo cycles of fission and fusion. Dynamin-related protein 1 (Drp1), a large GTPase of the dynamin superfamily, is the main mediator of mitochondrial fission. Like prototypical dynamin, Drp1 is composed of a mechanochemical core consisting of the GTPase, middle, and GTPase effector domain regions. In place of the pleckstrin homology domain in dynamin, however, Drp1 contains an unstructured variable domain, whose function is not yet fully resolved. Here, using time-resolved EM and rigorous statistical analyses, we establish the ability of full-length Drp1 to constrict lipid bilayers through a GTP hydrolysis-dependent mechanism. We also show the variable domain limits premature Drp1 assembly in solution and promotes membrane curvature. Furthermore, the mechanochemical core of Drp1, absent of the variable domain, is sufficient to mediate GTP hydrolysis-dependent membrane constriction.

Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells.

Adrenal neuroendocrine chromaffin cells receive excitatory synaptic input from the sympathetic nervous system and secrete hormones into the peripheral circulation. Under basal sympathetic tone, modest amounts of freely soluble catecholamine are selectively released through a restricted fusion pore formed between the secretory granule and the plasma membrane. Upon activation of the sympathoadrenal stress reflex, elevated stimulation drives fusion pore expansion, resulting in increased catecholamine secretion and facilitating release of copackaged peptide hormones. Thus regulated expansion of the secretory fusion pore is a control point for differential hormone release of the sympathoadrenal stress response. Previous work has shown that syndapin 1 deletion alters transmitter release and that the dynamin 1-syndapin 1 interaction is necessary for coupled endocytosis in neurons. Dynamin has also been shown to be involved in regulation of fusion pore expansion in neuroendocrine chromaffin cells through an activity-dependent association with syndapin. However, it is not known which syndapin isoform(s) contributes to pore dynamics in neuroendocrine cells. Nor is it known at what stage of the secretion process dynamin and syndapin associate to modulate pore expansion. Here we investigate the expression and localization of syndapin isoforms and determine which are involved in mediating fusion pore expansion. We show that all syndapin isoforms are expressed in the adrenal medulla. Mutation of the SH3 dynamin-binding domain of all syndapin isoforms shows that fusion pore expansion and catecholamine release are limited specifically by mutation of syndapin 3. The mutation also disrupts targeting of syndapin 3 to the cell periphery. Syndapin 3 exists in a persistent colocalized state with dynamin 1.

Ascl1a/Dkk/beta-catenin signaling pathway is necessary and glycogen synthase kinase-3beta inhibition is sufficient for zebrafish retina regeneration.

Key to successful retina regeneration in zebrafish are Müller glia (MG) that respond to retinal injury by dedifferentiating into a cycling population of retinal progenitors. Although recent studies have identified several genes involved in retina regeneration, the signaling mechanisms underlying injury-dependent MG proliferation have remained elusive. Here we report that canonical Wnt signaling controls the proliferation of MG-derived retinal progenitors. We found that injury-dependent induction of Ascl1a suppressed expression of the Wnt signaling inhibitor, Dkk, and induced expression of the Wnt ligand, Wnt4a. Genetic and pharmacological inhibition of Wnt signaling suppressed injury-dependent proliferation of MG-derived progenitors. Remarkably, in the uninjured retina, glycogen synthase kinase-3ß (GSK-3ß) inhibition was sufficient to stimulate MG dedifferentiation and the formation of multipotent retinal progenitors that were capable of differentiating into all major retinal cell types. Importantly, Ascl1a expression was found to contribute to the multipotential character of these progenitors. Our data suggest that Wnt signaling and GSK-3ß inhibition, in particular, are crucial for successful retina regeneration.

Differential curvature sensing and generating activities of dynamin isoforms provide opportunities for tissue-specific regulation.

Dynamin 1 (Dyn1) and Dyn2 are neuronal and ubiquitously expressed isoforms, respectively, of the multidomain GTPase required for clathrin-mediated endocytosis (CME). Although they are 79% identical, Dyn1 and Dyn2 are not fully functionally redundant. Through direct measurements of basal and assembly-stimulated GTPase activities, membrane binding, self-assembly, and membrane fission on planar and curved templates, we have shown that Dyn1 is an efficient curvature generator, whereas Dyn2 is primarily a curvature sensor. Using Dyn1/Dyn2 chimeras, we identified the lipid-binding pleckstrin homology domain as being responsible for the differential in vitro properties of these two isoforms. Remarkably, their in vitro activities were reversed by a single amino acid change in the membrane-binding variable loop 3. Reconstitution of KO mouse embryo fibroblasts showed that both the pleckstrin homology and the Pro/Arg-rich domains determine the differential abilities of these two isoforms to support CME. These domains are specific to classical dynamins and are involved in regulating their activity. Our findings reveal opportunities for fundamental differences in the regulation of Dyn1, which mediates rapid endocytosis at the synapse, vs. Dyn2, which regulates early and late events in CME in nonneuronal cells.

Allosteric control of dynamin-related protein 1 through a disordered C-terminal Short Linear Motif.

The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial and peroxisomal fission, but the regulatory mechanisms remain ambiguous. Here we find that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, surprisingly leading to the fission of model membranes in vitro. In vivo, the involvement of the native CT-SLiM is critical for productive mitochondrial and peroxisomal fission, as both deletion and non-native extension of the CT-SLiM severely impair their progression. Thus, contrary to prevailing models, Drp1-catalyzed membrane fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.