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, and their spatial and temporal coordination is crucial for efficient CME. Here, we show that the scaffold protein intersectin1 (ITSN1) promotes CME by organizing and stabilizing endocytic protein interaction networks. Live-cell imaging of genome-edited cells revealed that endogenously labeled ITSN1 is recruited during CME site stabilization and growth and that ITSN1 knockdown impairs endocytic protein recruitment during this stage.

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 protein Yap1801/Yap1802 (budding yeast AP180) as a key CME factor that promotes CME initiation in daughter cells during polarized growth, but how Yap1801/2 is 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 act as v-SNARES for exocytic vesicles arriving at the plasma membrane and are essential for polarized cell growth.

Myosin-I's motor and actin assembly activation activities are modular and separable in budding yeast clathrin-mediated endocytosis.

The myosin-Is, Myo3 and Myo5 in budding yeast, are implicated in force generation and actin assembly during clathrin-mediated endocytosis (CME). The myosin-Is have motor activity, bind the plasma membrane, and activate the Arp2/3 complex to promote branched actin assembly. We reveal that Myo5 's force-generating motor activity and nucleation-promoting factor (NPF) activity each must be coupled to membrane binding for successful CME.

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.

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.

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

The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, using quantitative live-cell microscopy in genome-edited Jurkat cells, we investigate the impact of Nef on clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment is associated with an increase in the recruitment and lifetime of the CME coat protein AP-2 and the late-arriving CME protein dynamin2.

Endocytic myosin-1 is a force-insensitive, power-generating motor.

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.

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.

Cik1 and Vik1 accessory proteins confer distinct functions to the kinesin-14 Kar3.

The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner.

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 actin's role in CME, is lacking.

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.

Endocytic myosin-1 is a force-insensitive, power-generating motor.

Unlabelled: 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.

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

Unlabelled: 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 actin’s role in CME, is lacking.

Membrane curvature as a signal to ensure robustness of diverse cellular processes.

An increasing corpus of research has demonstrated that membrane shape, generated either by the external environment of the cell or by intrinsic mechanisms such as cytokinesis and vesicle or organelle formation, is an important parameter in the control of diverse cellular processes. In this review we discuss recent findings that demonstrate how membrane curvature (from nanometer to micron length-scales) alters protein function. We describe an expanding toolkit for experimentally modulating membrane curvature to reveal effects on protein function, and discuss how membrane curvature - far from being a passive consequence of the physical environment and the internal protein activity of a cell - is an important signal that controls protein affinity and enzymatic activity to ensure robust forward progression of key processes within the cell.

Reconstitution of kinetochore motility 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 microtubules, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested .

Branched actin networks are organized for asymmetric force production during clathrin-mediated endocytosis in mammalian cells.

Actin assembly facilitates vesicle formation in several trafficking pathways, including clathrin-mediated endocytosis (CME). Interestingly, actin does not assemble at all CME sites in mammalian cells. How actin networks are organized with respect to mammalian CME sites and how assembly forces are harnessed, are not fully understood.

Induced nanoscale membrane curvature bypasses the essential endocytic function of clathrin.

During clathrin-mediated endocytosis (CME), flat plasma membrane is remodeled to produce nanometer-scale vesicles. The mechanisms underlying this remodeling are not completely understood. The ability of clathrin to bind membranes of distinct geometries casts uncertainty on its specific role in curvature generation/stabilization.

Mechanistic insights into actin force generation during vesicle formation 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 identified actin filament number, organization, and orientation during clathrin-mediated endocytosis in human SK-MEL-2 cells, showing that force generation is robust despite variance in network organization.

Load adaptation by endocytic actin networks.

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 has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension.

Unexplored Cdc42 functions at the budding yeast nucleus suggested by subcellular localization.

In budding yeast, the Rho-family GTPase Cdc42 has several functions that depend on its subcellular localization and the cell cycle stage. During bud formation, Cdc42 localizes to the plasma membrane at the bud tip and bud neck where it carries out functions in actin polymerization, spindle positioning, and exocytosis to ensure proper polarity development. Recent live-cell imaging analysis revealed a novel localization of Cdc42 to a discrete intracellular focus associated with the vacuole and nuclear envelope.

A preferred sequence for organelle inheritance 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.

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 nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they 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 intermembrane gap between neighboring cells.

Spatial regulation of clathrin-mediated endocytosis through position-dependent site maturation.

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, which cluster 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.