Structural basis for GTPase activity and conformational changes of the bacterial dynamin-like protein SynDLP.

SynDLP, a dynamin-like protein (DLP) encoded in the cyanobacterium Synechocystis sp. PCC 6803, has recently been identified to be structurally highly similar to eukaryotic dynamins. To elucidate structural changes during guanosine triphosphate (GTP) hydrolysis, we solved the cryoelectron microscopy (cryo-EM) structures of oligomeric full-length SynDLP after addition of guanosine diphosphate (GDP) at 4.1 Å and GTP at 3.6-Å resolution as well as a GMPPNP-bound dimer structure of a minimal G-domain construct of SynDLP at 3.8-Å resolution. In comparison with what has been seen in the previously resolved apo structure, we found that the G-domain is tilted upward relative to the stalk upon GTP hydrolysis and that the G-domain dimerizes via an additional extended dimerization domain not present in canonical G-domains. When incubated with lipid vesicles, we observed formation of irregular tubular SynDLP assemblies that interact with negatively charged lipids. Here, we provide the structural framework of a series of different functional SynDLP assembly states during GTP turnover.

Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4.

Eps15 (epidermal growth factor receptor pathway substrate 15) homology domain-containing proteins (EHDs) comprise a family of eukaryotic dynamin-related ATPases that participate in various endocytic membrane trafficking pathways. Dysregulation of EHDs function has been implicated in various diseases, including cancer. The lack of small molecule inhibitors which acutely target individual EHD members has hampered progress in dissecting their detailed cellular membrane trafficking pathways and their function during disease. Here, we established a Malachite green-based assay compatible with high throughput screening to monitor the liposome-stimulated ATPase of EHD4. In this way, we identified a drug-like molecule that inhibited EHD4's liposome-stimulated ATPase activity. Structure activity relationship (SAR) studies indicated sites of preferred substitutions for more potent inhibitor synthesis. Moreover, the assay optimization in this work can be applied to other dynamin family members showing a weak and liposome-dependent nucleotide hydrolysis activity.

Comparing Multifunctional Viral and Eukaryotic Proteins for Generating Scission Necks in Membranes.

Deterministic formation of membrane scission necks by protein machinery with multiplexed functions is critical in biology. A microbial example is M2 viroporin, a proton pump from the influenza A virus that is multiplexed with membrane remodeling activity to induce budding and scission in the host membrane during viral maturation. In comparison, the dynamin family constitutes a class of eukaryotic proteins implicated in mitochondrial fission, as well as various budding and endocytosis pathways. In the case of Dnm1, the mitochondrial fission protein in yeast, the membrane remodeling activity is multiplexed with mechanoenzyme activity to create fission necks. It is not clear why these functions are combined in these scission processes, which occur in drastically different compositions and solution conditions. In general, direct experimental access to changing neck sizes induced by individual proteins or peptide fragments is challenging due to the nanoscale dimensions and influence of thermal fluctuations. Here, we use a mechanical model to estimate the size of scission necks by leveraging small-angle X-ray scattering structural data of protein-lipid systems under different conditions. The influence of interfacial tension, lipid composition, and membrane budding morphology on the size of the induced scission necks is systematically investigated using our data and molecular dynamic simulations. We find that the M2 budding protein from the influenza A virus has robust pH-dependent membrane activity that induces nanoscopic necks within the range of spontaneous hemifission for a broad range of lipid compositions. In contrast, the sizes of scission necks generated by mitochondrial fission proteins strongly depend on lipid composition, which suggests a role for mechanical constriction.

Mesoscopic elasticity controls dynamin-driven fission of lipid tubules.

Mesoscale physics bridges the gap between the microscopic degrees of freedom of a system and its large-scale continuous behavior and highlights the role of a few key quantities in complex and multiscale phenomena, like dynamin-driven fission of lipid membranes. The dynamin protein wraps the neck formed during clathrin-mediated endocytosis, for instance, and constricts it until severing occurs. Although ubiquitous and fundamental for life, the cooperation between the GTP-consuming conformational changes within the protein and the full-scale response of the underlying lipid substrate is yet to be unraveled. In this work, we build an effective mesoscopic model from constriction to fission of lipid tubules based on continuum membrane elasticity and implicitly accounting for ratchet-like power strokes of dynamins. Localization of the fission event, the overall geometry, and the energy expenditure we predict comply with the major experimental findings. This bolsters the idea that a continuous picture emerges soon enough to relate dynamin polymerization length and membrane rigidity and tension with the optimal pathway to fission. We therefore suggest that dynamins found in in vivo processes may optimize their structure accordingly. Ultimately, we shed light on real-time conductance measurements available in literature and predict the fission time dependency on elastic parameters.

Cryo-EM structures of membrane-bound dynamin in a post-hydrolysis state primed for membrane fission.

Dynamin assembles as a helical polymer at the neck of budding endocytic vesicles, constricting the underlying membrane as it progresses through the GTPase cycle to sever vesicles from the plasma membrane. Although atomic models of the dynamin helical polymer bound to guanosine triphosphate (GTP) analogs define earlier stages of membrane constriction, there are no atomic models of the assembled state post-GTP hydrolysis. Here, we used cryo-EM methods to determine atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in a guanosine diphosphate (GDP)-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix with an inner lumen of 3.4 nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using the GTPase-defective dynamin K44A mutant and observed diverse dynamin helices, demonstrating that dynamin can accommodate a range of assembled complexes in cells that likely precede membrane fission.

The variable domain from dynamin-related protein 1 promotes liquid-liquid phase separation that enhances its interaction with cardiolipin-containing membranes.

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" important for regulation. For the mitochondrial fission dynamin, dynamin-related protein 1, a regulatory role for the variable domain (VD) is demonstrated by gain- and loss-of-function mutations, yet the basis for this is unclear. Here, the isolated VD is shown to be intrinsically disordered and undergo a cooperative transition in the stabilizing osmolyte trimethylamine N-oxide. However, the osmolyte-induced state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, which appears to promote phase separation. Since dynamin-related protein 1 is found assembled into discrete punctate structures on the mitochondrial surface, the inference from the present work is that these structures might arise from a condensed state involving the VD that may enable rapid tuning of mechanoenzyme assembly necessary for fission.

"Gearing" up for dynamin-catalyzed membrane fission.

Endocytic dynamins self-assemble into helical scaffolds and utilize energy from GTP hydrolysis to constrict and sever tubular membranous necks of budded endocytic intermediates. They bind the membrane using a pleckstrin-homology domain (PHD). The PHD is characterized by four unstructured loops, two of which partially insert into the membrane. Recent studies reveal that loop insertion lowers the bending rigidity of the membrane and that mutations in these two loops produce separable and opposite effects on the efficiency of dynamin-catalyzed membrane fission. Here, we review the current understanding of dynamin-catalyzed membrane fission and attempt to reconcile contrasting notions that have emerged from biochemical and cellular studies evaluating the role of the PHD in this process. We propose that two membrane-inserting loops act as "gears" that define the catalytic efficiency of the dynamin helical scaffold in membrane fission.

DHA alleviates high glucose-induced mitochondrial dysfunction in Oreochromis niloticus by inhibiting DRP1-mediated mitochondrial fission.

Dynamin-related protein 1 (DRP1) is a key regulator in the maintenance of mammalian glucose homeostasis, but the relevant information remains poorly understood on aquatic animals. In the study, DRP1 is formally described for the first time in Oreochromis niloticus. DRP1 encodes a peptide of 673 amino acid residues that contained three conserved domains: a GTPase domain, a dynamin middle domain and a dynamin GTPase effector domain. DRP1 transcripts are widely distributed in all of the detected seven organs/tissues, and the highest mRNA levels in brain. High-carbohydrate (45 %) fed fish showed a significant upregulation of liver DRP1 expression than that of control (30 %) group. Glucose administration upregulated liver DRP1 expression, with peak values observed at 1 h; then its expression returned to the basal value at 12 h. In the in vitro study, DRP1 over-expression significantly decreased mitochondrial abundance in hepatocytes. DHA significantly increased mitochondrial abundance, transcriptions of mitochondrial transcription factor A (TFAM) and mitofusin 1 and 2 (MFN1 and MFN2) and complex II and III activities of high glucose-treated hepatocyte, whereas the opposite was true for DRP1, mitochondrial fission factor (MFF) and fission (FIS) expression. Together, these findings illustrated that O. niloticus DRP1 is highly conserved, and it participated in glucose control of fish. DHA could alleviate high glucose-induced mitochondrial dysfunction of fish by inhibiting DRP1-mediated mitochondrial fission.

Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction.

Mitochondrial fission is critical for mitochondrial dynamics and homeostasis. The dynamin superfamily GTPase DRP1 is recruited by three functionally redundant receptors, MFF, MiD49, and MiD51, to mitochondria to drive fission. Here, we exploit high-content live-cell imaging to screen for mitochondrial fission inhibitors and have developed a covalent compound, mitochondrial division inhibitor (MIDI). MIDI treatment potently blocks mitochondrial fragmentation induced by mitochondrial toxins and restores mitochondrial morphology in fusion-defective cells carrying pathogenic mitofusin and OPA1 mutations. Mechanistically, MIDI does not affect DRP1 tetramerization nor DRP1 GTPase activity but does block DRP1 recruitment to mitochondria. Subsequent biochemical and cellular characterizations reveal an unexpected mechanism that MIDI targets DRP1 interaction with multiple receptors via covalent interaction with DRP1-C367. Taken together, beyond developing a potent mitochondrial fission inhibitor that profoundly impacts mitochondrial morphogenesis, our study establishes proof of concept for developing protein-protein interaction inhibitors targeting DRP1.

From dynamin related proteins structures and oligomers to membrane fusion mediated by mitofusins.

Mitochondria assemble in a highly dynamic network where interconnected tubules evolve in length and size through regulated cycles of fission and fusion of mitochondrial membranes thereby adapting to cellular needs. Mitochondrial fusion and fission processes are mediated by specific sets of mechano-chemical large GTPases that belong to the Dynamin-Related Proteins (DRPs) super family. DRPs bind to cognate membranes and auto-oligomerize to drive lipid bilayers remodeling in a nucleotide dependent manner. Although structural characterization and mechanisms of DRPs that mediate membrane fission are well established, the capacity of DRPs to mediate membrane fusion is only emerging. In this review, we discuss the distinct structures and mechanisms of DRPs that trigger the anchoring and fusion of biological membranes with a specific focus on mitofusins that are dedicated to the fusion of mitochondrial outer membranes. In particular, we will highlight oligomeric assemblies of distinct DRPs and confront their mode of action against existing models of mitofusins assemblies with emphasis on recent biochemical, structural and computational reports. As we will see, the literature brings valuable insights into the presumed macro-assemblies mitofusins may form during anchoring and fusion of mitochondrial outer membranes.

Mitochondrial dynamics and its impact on human health and diseases: inside the DRP1 blackbox.

Mitochondria are essential organelles that play a significant role in various cellular processes apart from providing energy in eukaryotic cells. An intricate link between mitochondrial structure and function is now unequivocally accepted. Several molecular players have been identified, which are important in maintaining the structure of the organelle. Dynamin-related protein 1 (DRP1) is one such conserved protein that is a vital regulator of mitochondrial dynamics. Multidisciplinary studies have helped elucidate the structure of the protein and its mechanism of action in great detail. Mutations in various domains of the protein have been identified that are associated with debilitating conditions in patients. The involvement of the protein in disease conditions such as neurodegeneration, cancer, and cardiovascular disorders is also gaining attention. The purpose of this review is to highlight recent findings on the role of DRP1 in human disease conditions and address its importance as a therapeutic target.

Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers.

The quantification of membrane-associated biomolecular interactions is crucial to our understanding of various cellular processes. State-of-the-art single-molecule approaches rely largely on the addition of fluorescent labels, which complicates the quantification of the involved stoichiometries and dynamics because of low temporal resolution and the inherent limitations associated with labeling efficiency, photoblinking and photobleaching. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Application of this method to the membrane remodeling GTPase, dynamin-1, reveals heterogeneous mixtures of dimer-based oligomers, oligomer-dependent mobilities, membrane affinities and (dis)association of individual complexes. These capabilities, together with assay-based advances for studying integral membrane proteins, will enable the elucidation of biomolecular mechanisms in and on lipid bilayers.

Pathogenic DNM1L Variant (1085G>A) Linked to Infantile Progressive Neurological Disorder: Evidence of Maternal Transmission by Germline Mosaicism and Influence of a Contemporary in cis Variant (1535T>C).

Mitochondria are dynamic organelles undergoing continuous fusion and fission with Drp1, encoded by the DNM1L gene, required for mitochondrial fragmentation. DNM1L dominant pathogenic variants lead to progressive neurological disorders with early exitus. Herein we report on the case of a boy affected by epileptic encephalopathy carrying two heterozygous variants (in cis) of the DNM1L gene: a pathogenic variant (PV) c.1085G>A (p.Gly362Asp) accompanied with a variant of unknown significance (VUS) c.1535T>C (p.Ile512Thr). Amplicon sequencing of the mother's DNA revealed the presence of the PV and VUS in 5% of cells, with the remaining cells presenting only VUS. Functional investigations performed on the patient and his mother's cells unveiled altered mitochondrial respiratory chain activities, network architecture and Ca2+ homeostasis as compared with healthy unrelated subjects' samples. Modelling Drp1 harbouring the two variants, separately or in combination, resulted in structural changes as compared with Wt protein. Considering the clinical history of the mother, PV transmission by a maternal germline mosaicism mechanism is proposed. Altered Drp1 function leads to changes in the mitochondrial structure and bioenergetics as well as in Ca2+ homeostasis. The novel VUS might be a modifier that synergistically worsens the phenotype when associated with the PV.

Quantification and demonstration of the collective constriction-by-ratchet mechanism in the dynamin molecular motor.

Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single-molecule Förster resonance energy transfer (smFRET) and found strong nucleotide-dependent conformational preferences. Integrating smFRET with molecular dynamics simulations allowed us to estimate the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GTPase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved.

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.

Mimicking human Drp1 disease-causing mutations in yeast Dnm1 reveals altered mitochondrial dynamics.

The dynamin-related protein 1 (Drp1) and its homologs in various eukaryotes are essential to maintain mitochondrial morphology and regulate mitochondrial division. Several mutations in different domains of Drp1 have been reported, which result in debilitating conditions. Four such disease-causing mutations of the middle domain of Drp1 were mimicked in the yeast dynamin-related GTPase (Dnm1) and were characterized in this study. Mitochondrial morphology and protein function were observed to be altered to a variable extent in cells expressing the mutated variants of Dnm1. Several aspects related to the protein such as punctate formation, localization to mitochondria, dynamic behavior and structure were analyzed by microscopy, biochemical studies and molecular dynamics simulations. Significant effects on the protein structure and function were observed in cells expressing A430D and G397D mutations. Overall, our data provide insight into the molecular and cellular alterations resulting from middle domain mutations in Dnm1.

Cardiolipin targets a dynamin-related protein to the nuclear membrane.

Dynamins are targeted to specific cellular membranes that they remodel via membrane fusion or fission. The molecular basis of conferring specificity to dynamins for their target membrane selection is not known. Here, we report a mechanism of nuclear membrane recruitment of Drp6, a dynamin member in Tetrahymena thermophila. Recruitment of Drp6 depends on a domain that binds to cardiolipin (CL)-rich bilayers. Consistent with this, nuclear localization of Drp6 was inhibited either by depleting cellular CL or by substituting a single amino acid residue that abolished Drp6 interactions with CL. Inhibition of CL synthesis, or perturbation in Drp6 recruitment to nuclear membrane, caused defects in the formation of new macronuclei post-conjugation. Taken together, our results elucidate a molecular basis of target membrane selection by a nuclear dynamin and establish the importance of a defined membrane-binding domain and its target lipid in facilitating nuclear expansion.

Function and regulation of the divisome for mitochondrial fission.

Mitochondria form dynamic networks in the cell that are balanced by the flux of iterative fusion and fission events of the organelles. It is now appreciated that mitochondrial fission also represents an end-point event in a signalling axis that allows cells to sense and respond to external cues. The fission process is orchestrated by membrane-associated adaptors, influenced by organellar and cytoskeletal interactions and ultimately executed by the dynamin-like GTPase DRP1. Here we invoke the framework of the 'mitochondrial divisome', which is conceptually and operationally similar to the bacterial cell-division machinery. We review the functional and regulatory aspects of the mitochondrial divisome and, within this framework, parse the core from the accessory machinery. In so doing, we transition from a phenomenological to a mechanistic understanding of the fission process.

Drp1-dependent mitochondrial fission in cardiovascular disease.

Mitochondria are highly dynamic organelles undergoing cycles of fusion and fission to modulate their morphology, distribution, and function, which are referred as 'mitochondrial dynamics'. Dynamin-related protein 1 (Drp1) is known as the major pro-fission protein whose activity is tightly regulated to clear the damaged mitochondria via mitophagy, ensuring a strict control over the intricate process of cellular and organ dynamics in heart. Various posttranslational modifications (PTMs) of Drp1 have been identified including phosphorylation, SUMOylation, palmitoylation, ubiquitination, S-nitrosylation, and O-GlcNAcylation, which implicate a role in the regulation of mitochondrial dynamics. An intact mitochondrial homeostasis is critical for heart to fuel contractile function and cardiomyocyte metabolism, while defects in mitochondrial dynamics constitute an essential part of the pathophysiology underlying various cardiovascular diseases (CVDs). In this review, we summarize current knowledge on the critical role of Drp1 in the pathogenesis of CVDs including endothelial dysfunction, smooth muscle remodeling, cardiac hypertrophy, pulmonary arterial hypertension, myocardial ischemia-reperfusion, and myocardial infarction. We also highlight how the targeting of Drp1 could potentially contribute to CVDs treatments.

GFP fluorescence tagging alters dynamin-related protein 1 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission.

Green fluorescent protein (GFP)-tagging is the prevalent strategy to monitor protein dynamics in living cells. However, the consequences of appending the bulky GFP moiety to the protein of interest are rarely investigated. Here, using a powerful combination of quantitative fluorescence spectroscopic and imaging techniques, we have examined the oligomerization dynamics of the GFP-tagged mitochondrial fission GTPase dynamin-related protein 1 (Drp1) both in vitro and in vivo. We find that GFP-tagged Drp1 exhibits impaired oligomerization equilibria in solution that corresponds to a greatly diminished cooperative GTPase activity in comparison to native Drp1. Consequently, GFP-tagged Drp1 constitutes aberrantly stable, GTP-resistant supramolecular assemblies both in vitro and in vivo, neither of which reflects a more dynamic native Drp1 oligomerization state. Indeed, GFP-tagged Drp1 is detected more frequently per unit length over mitochondria in Drp1-null mouse embryonic fibroblasts (MEFs) compared to wild-type (wt) MEFs, indicating that the drastically reduced GTP turnover restricts oligomer disassembly from the mitochondrial surface relative to mixed oligomers comprising native and GFP-tagged Drp1. Yet, GFP-tagged Drp1 retains the capacity to mediate membrane constriction in vitro and mitochondrial division in vivo. These findings suggest that instead of robust assembly-disassembly dynamics, persistent Drp1 higher-order oligomerization over membranes is sufficient for mitochondrial fission.