Biology subjects

Drubin, D. G.

Publications and source records attributed to Drubin, D. G..

25 records · Page 2Linked to original sources

bioRxiv2020

Adaptive actin organization counteracts elevated membrane tension to ensure robust endocytosis

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load, as has been observed in lamellipodia, has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feedback on assembly. By manipulating membrane tension and Arp2/3 complex activity we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions.

cell biology↗

bioRxiv2019

Cargo licenses maturation of stalled endocytic sites through a regulatory checkpoint

During clathrin-mediated endocytosis (CME), over 50 different proteins assemble on the plasma membrane to reshape it into a cargo-laden vesicle. It has long been assumed that cargo triggers local CME site assembly in Saccharomyces cerevisiae based on the discovery that cortical actin patches clustered near exocytic sites are CME sites. Quantitative imaging data reported here lead to a radically different view of which CME steps are regulated and which steps are deterministic. We quantitatively and spatially describe progression through the CME pathway and pinpoint a cargo-sensitive regulatory transition point that governs progression from the initiation phase of CME to the internalization phase. Thus, site maturation, rather than site initiation, accounts for the previously observed polarized distribution of actin patches in this organism. While previous studies suggested that cargo ensures its own internalization by regulating either CME initiation rates or frequency of abortive events, our data instead identify maturation through a checkpoint in the pathway as the cargo-sensitive step. SummaryPedersen, Hassinger, et al. investigate steps of the clathrin-mediated endocytosis pathway that are subject to regulation. They report position-dependent differences in endocytic site maturation rates in polarized cells and suggest that cargo controls endocytic internalization through tuning site maturation rather than site initiation.

cell biology↗

bioRxiv2019

Cdc42 GTPase regulates ESCRTs in nuclear envelope sealing and ER remodeling

Small GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none involved in the maintenance of nuclear envelope integrity or endoplasmic reticulum (ER) morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission, and by genetic analysis, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture.\n\nSummaryThe small Rho GTPase Cdc42 is a well-known regulator of cytoskeletal rearrangement and polarity development in all eukaryotic cell types. Here, Lu and Drubin report the serendipitous discovery of a novel Cdc42-ESCRT-nuclear envelope/endoplasmic reticulum connection.

cell biology↗

bioRxiv2019

Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features

AbstractConserved proteins drive clathrin-mediated endocytosis (CME), which universally involves a burst of actin assembly. To gain fundamental mechanistic insights into this process, a side-by-side quantitative comparison of CME was performed on two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae are more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast than in budding yeast. In both yeasts, Arp2/3 complex activation drives membrane invagination when triggered by the accumulation of [~]70 WASP molecules. In contrast to budding yeast, WASP-mediated actin nucleation activity plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, though two-zone actin assembly is a fission yeast-specific mechanism, which is not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations and have broad implications that extend from yeast to humans.

cell biology↗

bioRxiv2019

Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

Force generation due to actin assembly is a fundamental aspect of membrane sculpting for many essential processes. In this work, we use a multiscale computational model constrained by experimental measurements to show that a minimal branched actin network is sufficient to internalize endocytic pits against physiological membrane tension. A parameter sweep identified the number of Arp2/3 complexes as particularly important for robust internalization, which prompted the development of a molecule-counting method in live mammalian cells. Using this method, we found that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Our simulations also revealed that actin networks self-organize in a radial branched array with barbed filament ends oriented to grow toward the base of the pit, and that the distribution of linker proteins around the endocytic pit is critical for this organization. Surprisingly, our model predicted that long actin filaments bend from their attachment sites in the coat to the base of the pit and store elastic energy that can be harnessed to drive endocytosis. This prediction was validated using cryo-electron tomography on cells, which revealed the presence of bent actin filaments along the endocytic site. Furthermore, we predict that under elevated membrane tension, the self-organized actin network directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, our study reveals that spatially constrained actin filament assembly utilizes an adaptive mechanism that enables endocytosis under varying physical constraints.

cell biology↗

bioRxiv2019

Simultaneous 3D super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for cell imaging

Correlating data from different microscopy techniques holds the potential to discover new facets of signaling events in cellular biology. Here we report for the first time a hardware set-up capable of achieving simultaneous imaging of spatially correlated super-resolution fluorescence microscopy and atomic force microscopy, a feat only obtained until now by fluorescence microscopy set-ups with spatial resolution restricted to the Abbe resolution limit. We hereby remove the need to perform independent measurement and subsequent data averaging required to eliminate cell-to-cell variation in observed signals. We detail system integration, demonstrate system performance and report imaging of sub-resolution fluorescent beads and genome-engineered human bone osteosarcoma epithelial cells.

cell biology↗

bioRxiv2018

Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.\n\nOne Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

cell biology↗