David G. Drubin

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. Statement of Significance: Clathrin-mediated endocytosis is the essential pathway through which a patch of plasma membrane deforms into a vesicle in all eukaryotic cells. Recent experimental studies suggest that cytoplasmic components of the endocytic machinery condense into a liquid-like droplet on the membrane prior to internalization, raising questions about the droplet’s biological function. Using complementary numerical and analytical modeling approaches, we show that an endocytic droplet can regulate the pathway via capillary force. The droplet volume sets the strength of this force, providing the cell with a handle to switch from stalling to driving membrane invagination. While the biological role of liquid condensates is typically attributed to organizing biochemistry, this work demonstrates a novel purely mechanical function.

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 . Short Summary: Quantitative in vivo titration shows that eight yeast endocytic proteins form distinct, enlarged assemblies. Reversible phosphorylation dynamically tunes assembly thresholds, orchestrating a two-stage condensate reorganization from initiation to maturation during CME.

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 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 superresolution microscopy of genome-edited human induced pluripotent stem cells 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 increase actin assembly and motor activity, 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.

Clathrin-mediated endocytosis (CME) is an essential, highly conserved process in eukaryotic cells that facilitates the internalization of plasma membrane components, transmembrane proteins and extracellular nutrients. This complex pathway involves the concerted assembly and disassembly of many different proteins at the plasma membrane. Budding yeast has served as a powerful model for dissecting CME through combined genetic, biochemical, quantitative imaging and mathematical approaches. In this Cell Science at a Glance article, we integrate decades of quantitative data to generate a three-dimensional molecular animation depicting the full progression of CME in budding yeast (Movie 1). The animation and accompanying poster capture the spatial and temporal dynamics of key protein players. In addition, we highlight recent advances in understanding of the condensation of endocytic proteins into distinct sites and the organization of actin networks that generate the forces necessary to deform and internalize the membrane against the high internal turgor pressure of the budding yeast cell.

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. Significance: For 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.

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 for biological processes sensitive to altered protein stoichiometry, such as clathrin-mediated endocytosis (CME), editing genomes of metazoan cells incurs a significant time cost compared with 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 spatial 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 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.

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 Statement Cell fusion can be used to generate new multi-colored genome-edited cell lines, which can be clonally expanded. In 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. Multiple 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.

Understanding biological systems requires observing features and processes across vast spatial and temporal scales, spanning nanometers to centimeters and milliseconds to days, often using multiple imaging modalities within complex native microenvironments. Yet, achieving this comprehensive view is challenging because microscopes optimized for specific tasks typically lack versatility due to inherent optical and sample handling trade-offs, and frequently suffer performance degradation from sample-induced optical aberrations in multicellular contexts. Here, we present MOSAIC, a reconfigurable microscope that integrates multiple advanced imaging techniques including light-sheet, label-free, super-resolution, and multi-photon, all equipped with adaptive optics. MOSAIC enables non-invasive imaging of subcellular dynamics in both cultured cells and live multicellular organisms, nanoscale mapping of molecular architectures across millimeter-scale expanded tissues, and structural/functional neural imaging within live mice. MOSAIC facilitates correlative studies across biological scales within the same specimen, providing an integrated platform for broad biological investigation.

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-I’s 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 Statement: Type-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. The 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. These 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.