Biology subjects

Drubin, D. G.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanical regulation of endocytosis by protein droplet capillary forces

Clathrin-mediated endocytosis (CME) is the primary pathway for internalization of extracellular and membrane cargo in eukaryotic cells. It is characterized by a patch of the plasma membrane invaginating and pinching off to generate a cytoplasmic vesicle. Recently, experimental evidence has begun to accumulate in support of proteins assembling into liquid-like condensates at nascent CME sites, but potential functional consequences are not understood. Given that condensate capillary forces can influence membrane bending, we constructed complementary analytical and numerical model systems to probe the mechanical influence of a protein droplet on endocytosis. Model evaluations and analysis reveal that a droplet can regulate the progression of endocytosis by stalling invagination until the droplet grows past a threshold size. After the droplet-mediated brake is released, the droplet provides a force that can drive the membrane bud toward scission. Thus, we propose a ``mechanical timer'' mechanism as a function for protein phase separation in cellular membrane trafficking.

biophysics↗

bioRxiv2026

Phosphoregulation of hierarchical protein condensate formation during clathrin-mediated endocytosis

The sequential assembly of over 50 proteins during clathrin-mediated endocytosis (CME) is well documented in yeast. However, the mechanisms governing this ordered recruitment remain poorly understood. Biomolecular condensation is proposed to orchestrate CME, but defining biophysical parameters in vivo remains challenging. Here, we developed a quantitative in vivo titration strategy to systematically determine CME protein assembly profiles. Beyond the previously reported Ede1 (yeast Eps15), seven additional proteins formed enlarged, concentration-dependent assemblies with distinct biophysical properties. Single-cell profiling revealed two biophysical classes among these eight proteins: homeostatic buffers with defined saturation boundaries and continuous assemblers that scale linearly. Acting as scaffolds and clients, these conserved proteins form an interconnected network of kinases, phosphatase recruiters, and substrates. Within this network, reversible phosphorylation modulates assembly stability to drive recruitment transitions. Collectively, this work establishes a two-stage model of protein condensation that drives CME progression from initiation to maturation and offers a general framework for how protein phosphorylation dynamically modulates biomolecular condensation in vivo.

cell biology↗

bioRxiv2025

Class-I myosin responds to change in membrane tension during clathrin-mediated endocytosis in human induced pluripotent stem cells

Clathrin-mediated endocytosis (CME) is an essential cellular process that needs to operate efficiently across a wide range of conditions. Internalization of the endocytic site involves forces generated by membrane-bound proteins and Arp2/3-mediated branched actin filament assembly to bend the plasma membrane (PM) from flat to omega-shaped. In mammalian CME, the requirement for a branched actin filament network varies depending on cell type and differences in membrane tension. However, how the actin network adapts to changes in load in order to ensure robustness of this process over a range of membrane tensions is not understood. Here, we combine live-cell imaging and super-resolution microscopy of genome-edited human induced pluripotent stem cells (hiPSCs) to investigate the role of the mammalian class-I myosin, Myosin1E (Myo1E), in load adaptation. Under normal conditions, sites that recruit Myo1E are rare and exhibit slow CME dynamics. However, as membrane tension increases and CME dynamics are slowed globally, Myo1E is recruited to more sites, likely to assemble more branched actin, resulting in increased force generation to rescue stalled sites and promote internalization. Loss of Myo1E results in increased Arp2/3 complex lifetime at CME sites under normal conditions, and at high membrane tension, these sites fail to recruit as many Arp2/3 molecules. We propose that Myo1E is recruited to CME sites that have stalled due to increased membrane tension, where it helps build a more effective branched actin network by generating force through motor activity and recruiting additional Arp2/3 complexes to rescue stalled sites. SignificanceFor mammalian cells to internalize extracellular cargo via clathrin-mediated endocytosis (CME), specific regions of the plasma membrane (PM) must bend from flat to inwardly curved, a process that requires force-generating proteins. One key component in generating this force during CME is the branched actin network, in which actin filaments polymerize against the plasma membrane. When PM tension increases, more force is required to generate curvature, prompting the assembly of actin and actin associated proteins to aid the process. We demonstrate that the class-I myosin motor, Myosin1E (Myo1E), becomes increasingly crucial as membrane tension rises, presumably to build a more effective branched actin network to facilitate internalization of slowed sites.

cell biology↗

bioRxiv2025

Harnessing fusion of genome-edited human stem cells to rapidly screen for novel protein functions in vivo

Genome editing has enabled the integration of fluorescent protein coding sequences into genomes, resulting in expression of in-frame fusion proteins under the control of their natural gene regulatory sequences. While this technique overcomes the well-documented artifacts associated with gene overexpression, editing genomes of metazoan cells incurs a significant time cost compared to simpler organisms, such as yeast. Editing two or more genes to express multiple fluorescent fusion proteins in a single cell line has proven to be a powerful strategy for uncovering spatio-dynamic, and therefore functional, relationships among different proteins, but it can take many months to edit each gene within the same cell line. Here, by utilizing cell fusions, we quickly generated cells expressing pairwise permutations of fluorescent fusion proteins in genome-edited human cells to reveal previously undetected protein-organelle interactions. We fused human induced pluripotent stem cells (hiPSCs) that express in-frame fusions of clathrin-mediated endocytosis (CME) and actin cytoskeleton proteins with hiPSCs that express fluorescently tagged organelle markers, uncovering novel interactions between CME proteins, branched actin filament networks, and lysosomes. Significance StatementO_LICell fusion can be used to generate new multi-colored genome-edited cell lines, which can be clonally expanded. C_LIO_LIIn combination with genome-edited fluorescent cell line libraries such as the collection from the Allen Institute for Cell Science, cell fusion can be used as a screening tool to look for novel protein localization on a variety of intracellular structures. C_LIO_LIMultiple endocytic proteins localize to the surface of lysosomes in healthy, non-stressed cells, and these proteins exhibit a broad range of behaviors on the lysosome surface. C_LI

cell biology↗

bioRxiv2025

Force-insensitive myosin-I enhances endocytosis robustness through actin network-scale collective ratcheting

Force production by Type-I myosins influences endocytic progression in many cell types. Since different myosin-I isoforms exhibit distinct force-dependent kinetic properties, it is important to investigate how these properties affect endocytic outcomes, and the mechanisms through which myosin-I contributes to endocytosis. To this end, we adapted our agent-based simulations of endocytic actin networks and incorporated nonprocessive, single-headed myosin motors at the base of the endocytic pit. We varied the unbinding rate and the force dependence of myosin unbinding. Our results revealed that the inclusion of myosin motors facilitated endocytic internalization, but only under kinetic regimes with rapid and less force-sensitive unbinding. Conversely, slow or strongly force-dependent unbinding impeded endocytic progression. As membrane tension increased, the boundary between assistive and inhibitory phases shifted, allowing the myosins to assist over larger regions of the kinetic landscape. Myosin-Is contribution to internalization could not be explained by direct force transduction or increased actin assembly. Instead, the myosins collectively bolstered the robustness of internalization by limiting pit retraction. Significance StatementO_LIType-I myosins with varying force sensitivity levels participate in different membrane deformation pathways, but the mechanistic link between molecular biophysical properties and cellular function remains poorly understood. C_LIO_LIThe authors analyze a computational model of endocytosis with type-I myosins and find that myosins with lower force sensitivity assist endocytosis by reducing backsliding along the internalization trajectory, while myosins with higher force sensitivity stall endocytosis by sequestering actin in non-productive orientations. C_LIO_LIThese results introduce a new perspective on the function of type-I myosins in membrane reshaping: as a collective emergent property rather than the sum of individual force-generating motors. C_LI

biophysics↗

bioRxiv2024

Polarized exocytosis and anionic phospholipid species implicated in the initiation of clathrin-mediated endocytosis

Understanding of the mechanisms that initiate clathrin-mediated endocytosis (CME) is incomplete. Recent studies in budding yeast identified the endocytic adaptor proteins Yap1801/Yap1802 (budding yeast AP180) as key CME factors that promote CME initiation in daughter cells during polarized growth, but how Yap1801/2 are recruited preferentially to the plasma membrane of daughter cells is not clear. The only known cargos for Yap1801/2 in yeast are the synaptobrevins Snc1 and Snc2, which serve as v-SNARES for exocytic vesicles reaching the plasma membrane and are crucial for polarized cell growth. In this study, we examine the spatiotemporal dynamics of functional, fluorescent protein-tagged Snc2 expressed from its endogenous locus and provide evidence that, along with anionic phospholipids, Snc2 specifically recruits Yap1802 to growing daughter cells. This protein-protein interaction creates a direct link between polarized secretion and CME and has further implications in CME initiation.

cell biology↗

bioRxiv2024

Intersectin1 promotes clathrin-mediated endocytosis by organizing and stabilizing endocytic protein interaction networks

During clathrin-mediated endocytosis (CME), dozens of proteins are recruited to nascent CME sites on the plasma membrane. Coordination of endocytic protein recruitment in time and space is important for efficient CME. Here, we show that the multivalent scaffold protein intersectin1 (ITSN1) promotes CME by organizing and stabilizing endocytic protein interaction networks. By live-cell imaging of genome-edited cells, we observed that endogenously labeled ITSN1 is recruited to CME sites shortly after they begin to assemble. Knocking down ITSN1 impaired endocytic protein recruitment during the stabilization stage of CME site assembly. Artificially locating ITSN1 to the mitochondria surface was sufficient to assemble puncta consisting of CME initiation proteins, including EPS15, FCHO, adaptor proteins, the AP2 complex and epsin1 (EPN1), and the vesicle scission GTPase dynamin2 (DNM2). ITSN1 can form puncta and recruit DNM2 independently of EPS15/FCHO or EPN1. Our work redefines ITSN1s primary endocytic role as organizing and stabilizing the CME protein interaction networks rather than a previously suggested role in initiation and provides new insights into the multi-step and multi-zone organization of CME site assembly.

cell biology↗

bioRxiv2024

A Role for Cross-linking Proteins in Actin Filament Network Organization and Force Generation

The high turgor pressure across the plasma membrane of yeasts creates a requirement for substantial force production by actin polymerization and myosin motor activity for clathrin-mediated endocytosis (CME). Endocytic internalization is severely impeded in the absence of fimbrin, an actin filament crosslinking protein called Sac6 in budding yeast. Here, we combine live-cell imaging and mathematical modeling to gain new insights into the role of actin filament crosslinking proteins in force generation. Genetic manipulation showed that CME sites with more crosslinking proteins are more effective at internalization under high load. Simulations of an experimentally constrained, agent- based mathematical model recapitulate the result that endocytic networks with more double-bound fimbrin molecules internalize the plasma membrane against elevated turgor pressure more effectively. Networks with large numbers of crosslinks also have more growing actin filament barbed ends at the plasma membrane, where the addition of new actin monomers contributes to force generation and vesicle internalization. Our results provide a richer understanding of the crucial role played by actin filament crosslinking proteins during actin network force generation, highlighting the contribution of these proteins to the self-organization of the actin filament network and force generation under increased load.

cell biology↗

bioRxiv2023

Interdependence of a microtubule polymerase and a motor protein in establishment of kinetochore end-on attachments

Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side-bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. Spatial and temporal constraints hinder observation of these events in live cells. Therefore, we used our previously developed reconstitution assay to observe dynamics of kinetochores, the yeast kinesin-8, Kip3, and the microtubule polymerase, Stu2, in lysates prepared from metaphase-arrested budding yeast, Saccharomyces cerevisiae. Using total internal reflection fluorescence (TIRF) microscopy to observe kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, motility was shown to be dependent on both Kip3, as we reported previously, and Stu2. These proteins were shown to have distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore-microtubule attachment.

cell biology↗

bioRxiv2023

Kinetic investigation reveals an HIV-1 Nef-dependent increase in AP-2 recruitment and productivity at endocytic sites

Lentiviruses express non-enzymatic accessory proteins whose function is to subvert cellular machinery in the infected host. The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, we investigate the interaction between Nef and clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells, using quantitative live-cell microscopy in genome-edited Jurkat cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment correlates with an increase in the recruitment and lifetime of CME coat protein AP-2 and late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2, suggesting that Nef recruitment to CME sites promotes CME site maturation to ensure high efficiency in host protein downregulation.

cell biology↗

bioRxiv2023

An endocytic myosin essential for plasma membrane invagination powers motility against resistance

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated [~]10-fold by phosphorylation, and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than like that of slow anchoring myosin-1s found on endosomal membranes. We therefore propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells. SummaryPedersen, Snoberger et al. measure the force-sensitivity of the yeast endocytic the myosin-1 called Myo5 and find that it is more likely to generate power than to serve as a force-sensitive anchor in cells. Implications for Myo5s role in clathrin-mediated endocytosis are discussed.

cell biology↗

bioRxiv2023

Self-organizing actin networks drive sequential endocytic protein recruitment and vesicle release on synthetic lipid bilayers

Forces generated by actin assembly assist membrane invagination during clathrin-mediated endocytosis (CME). The sequential recruitment of core endocytic proteins and regulatory proteins, and assembly of the actin network, are well documented in live cells and are highly conserved from yeasts to humans. However, understanding of CME protein self-organization, as well as the biochemical and mechanical principles that underlie actins role in CME, is lacking. Here, we show that supported lipid bilayers coated with purified yeast WASP, an endocytic actin assembly regulator, and incubated in cytoplasmic yeast extracts, recruit downstream endocytic proteins and assemble actin tails. Time-lapse imaging of WASP-coated bilayers revealed sequential recruitment of proteins from different endocytic modules, faithfully replicating in vivo behavior. Reconstituted actin networks assemble in a WASP-dependent manner and deform lipid bilayers, as seen by electron microscopy. Time-lapse imaging revealed that vesicles are released from the lipid bilayers with a burst of actin assembly. Actin networks pushing on membranes have previously been reconstituted; here, we have reconstituted a biologically important variation of these actin networks that self-organize on bilayers and produce pulling forces sufficient to bud off membrane vesicles. We propose that actin-driven vesicle generation may represent an ancient evolutionary precursor to diverse vesicle forming processes adapted for a wide array of cellular environments and applications. Significance StatementActin filament assembly participates in many vesicle-forming processes. However, the underlying principles for how assembly is initiated and organized to effectively harness assembly forces remain elusive. To address this gap, we report a novel reconstitution of actin-driven vesicle release from supported lipid bilayers. Using real-time imaging, we observe sequential recruitment of endocytic proteins and, following a burst of actin assembly, vesicle release from bilayers. Given the absence of cargo or upstream endocytic regulatory proteins on the bilayers, and the participation of actin in many vesicle-forming processes, we posit that this mode of vesicle formation represents an early evolutionary precursor for multiple trafficking pathways. We expect that this assay will be of great use for future investigations of actin-mediated vesicle-forming processes.

cell biology↗

bioRxiv2022

Reconstitution of Kinetochore and Microtubule Dynamics Reveals a Role for a Kinesin-8 in Establishing End-on Attachments

During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between endogenously tagged tubulin, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae. Using both TIRF microscopy and cryo-correlative light microscopy and electron tomography, we successfully reconstituted microtubule-bound, intact kinetochores. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.

cell biology↗

bioRxiv2021

Asymmetric Arp2/3-mediated actin assembly facilitates clathrin-mediated endocytosis at stalled sites in genome-edited human stem cells.

Actin assembly facilitates vesicle formation in several trafficking pathways. Clathrin-mediated endocytosis (CME) shows elevated actin assembly dependence under high membrane tension. Why actin assembly at CME sites occurs heterogeneously even within the same cell, and how assembly forces are harnessed, are not fully understood. Here, endocytic dynamics, actin presence, and geometry of CME proteins from three different functional modules, were analyzed using three-dimensional (3D) super-resolution microscopy, live-cell imaging, and machine-learning-based computation. When hundreds of CME events were compared, sites with actin assembly showed a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. N-WASP is recruited to one side of CME sites where it is positioned to stimulate asymmetric actin assembly. We propose that asymmetric actin assembly rescues stalled CME sites by pulling vesicles into the cell much like a bottle opener pulls off a bottle cap.

cell biology↗

bioRxiv2021

Induced Membrane Curvature Bypasses Clathrin's Essential Endocytic Function

During membrane trafficking, flat membrane is rapidly remodeled to produce nanometer-scale vesicles. The mechanisms underlying this shape change are not completely understood, but coat proteins such as clathrin are implicated. Clathrins ability to bind to membranes of many different geometries casts uncertainty on its specific role in curvature generation and stabilization. Here, we used nanopatterning to produce substrates of ideal optical properties for live-cell imaging, with U-shaped features that bend a cells ventral plasma membrane into shapes characteristic of the energetically-unfavorable intermediate of clathrin- mediated endocytosis (CME). This induced plasma membrane curvature recruits the endocytic machinery, promoting productive endocytosis. Upon clathrin or AP2 disruption, CME sites on flat substrates are diminished. However, induced curvature rescues the localization, turnover and transferrin cargo uptake activities of CME sites after clathrin, but not AP2, disruption. These data establish that clathrins essential function during CME is to facilitate the evolution of membrane curvature rather than to scaffold CME protein recruitment. SummaryCail et al. demonstrate that induced nanoscale membrane curvature recruits endocytic sites and produces vesicles in cells lacking the coat protein clathrin, while the adaptor protein AP2 is still required, showing that clathrins essential function is to facilitate curvature development of the nascent vesicle.

cell biology↗

bioRxiv2021

Actin force generation in vesicle formation: mechanistic insights from cryo-electron tomography

Actin assembly provides force for a multitude of cellular processes. Compared to actin assembly- based force production during cell migration, relatively little is understood about how actin assembly generates pulling forces for vesicle formation. Here, cryo-electron tomography revealed actin filament number, organization, and orientation during clathrin-mediated endocytosis in human cells, showing that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating a measured branch angle indicate that sufficient force to drive membrane internalization is generated through polymerization, and that assembly is triggered from [~]4 founding "mother" filaments, consistent with tomography data. Hip1R actin filament anchoring points are present along the entire endocytic invagination, where simulations show that it is key to pulling force generation, and along the neck, where it targets filament growth and makes internalization more robust. Actin cytoskeleton organization described here allowed direct translation of structure to mechanism with broad implications for other actin-driven processes. Highlights- Filament anchorage points are key to pulling force generation and efficiency. - Native state description of CME-associated actin force-producing networks. - Branched actin filament assembly is triggered from multiple mother filaments. - Actin force production is robust despite considerable network variability.

cell biology↗

bioRxiv2021

An organelle inheritance pathway during polarized cell growth

Some organelles cannot be synthesized anew, so they are segregated into daughter cells during cell division. In Saccharomyces cerevisiae, daughter cells bud from mother cells and are populated by organelles inherited from the mothers. To determine whether this organelle inheritance occurs in a stereotyped manner, we tracked organelles using fluorescence microscopy. We describe a program for organelle inheritance in budding yeast. The cortical endoplasmic reticulum (ER) and peroxisomes are inherited concomitant with bud emergence. Next, vacuoles are inherited in small buds, followed closely by mitochondria. Finally, the nucleus and perinuclear ER are inherited when buds have nearly reached their maximal size. Because organelle inheritance timing correlates with bud morphology, which is coupled to the cell cycle, we tested whether organelle inheritance order is controlled by the cell cycle. By arresting cell cycle progression but allowing continued bud growth, we determined that organelle inheritance still occurs without cell cycle progression past S-phase, and that the general inheritance order is maintained. Thus, organelle inheritance follows a preferred order during polarized cell division, but it is not controlled exclusively by cell cycle signaling. Summary statementOrganelles are interconnected by contact sites, but they must be inherited from mother cells into buds during budding yeast mitosis. We report that this process occurs in a preferred sequence.

cell biology↗

bioRxiv2020

Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion

Fusion-associated small transmembrane (FAST) proteins are a diverse family of non-structural viral proteins that, once expressed on the plasma membrane of infected cells, drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the inter-membrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tails of p22 and p14 can be exchanging to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we replace p22s branched actin nucleator, N-WASP, with the parallel filament nucleator, formin, its ability to drive fusion is maintained, indicating that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

cell biology↗