Nuclei as mechanical bumpers during epithelial remodeling.

The morphogenesis of developing tissues relies on extensive cellular rearrangements in shape, position, and identity. A key process in reshaping tissues is cell intercalation-driven elongation, where epithelial cells align and intercalate along a common axis. Typically, analyses focus on how peripheral cortical forces influence cell shape changes.

Replisome loading reduces chromatin motion independent of DNA synthesis.

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged proliferating cell nuclear antigen (PCNA), a cell cycle marker and core component of the DNA replication machinery.

Interface extension is a continuum property suggesting a linkage between AP contractile and DV lengthening processes.

In the early embryo, the elongation of the anterior-posterior (AP) body axis is driven by cell intercalation in the germband epithelium. Neighboring cells intercalate through the contraction of AP interfaces (between AP neighbors) into higher-order vertices, which then resolve through the extension of new dorsal-ventral (DV) interfaces (between DV neighbors). Although interface contraction has been extensively studied, less is known about how new interfaces are established.

A PtdIns(3,4,5)P dispersal switch engages cell ratcheting at specific cell surfaces.

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating contractile forces before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be ratcheting mechanisms that drive processive transformations in cell shape. Previous work has shown that force generation is coordinated with endocytic remodeling; however, how ratcheting becomes engaged at specific cell surfaces remains unclear.

Vascular Aging in the Invertebrate Chordate, .

Vascular diseases affect over 1 billion people worldwide and are highly prevalent among the elderly, due to a progressive deterioration of the structure of vascular cells. Most of our understanding of these age-related cellular changes comes from studies on human cell lines. Further studies of the mechanisms underlying vascular aging are needed to provide insight into the pathobiology of age-associated vascular diseases, but are difficult to carry out on vertebrate model organisms.

Characterizing the cellular architecture of dynamically remodeling vascular tissue using 3-D image analysis and virtual reconstruction.

Epithelial tubules form critical structures in lung, kidney, and vascular tissues. However, the processes that control their morphogenesis and physiological expansion and contraction are not well understood. Here we examine the dynamic remodeling of epithelial tubes in vivo using a novel model system: the extracorporeal vasculature of , in which the disruption of the basement membrane triggers rapid, massive vascular retraction without loss of barrier function.

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway.

Phosphoinositides play crucial roles in intracellular membrane dynamics and cell signaling, with phosphatidylinositol (PI) 3-phosphates being the predominant phosphoinositide lipids at endosomes and lysosomes, whereas PI 4-phosphates, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P), are enriched at the cell surface including sites of endocytosis. How PI 4-phosphates and PI 3-phosphates are dynamically interconverted within the endocytic pathway and how this is controlled in space and time remains poorly understood. Here, combining live imaging, genome engineering, and acute chemical and genetic manipulations, we found that local synthesis of PI(3,4)P by phosphatidylinositol 3-kinase C2α at plasma membrane clathrin-coated pits is spatially segregated from its hydrolysis by the PI(3,4)P-specific inositol polyphosphate 4-phosphatase 4A (INPP4A).

Nanoscale coupling of endocytic pit growth and stability.

Clathrin-mediated endocytosis, an essential process for plasma membrane homeostasis and cell signaling, is characterized by stunning heterogeneity in the size and lifetime of clathrin-coated endocytic pits (CCPs). If and how CCP growth and lifetime are coupled and how this relates to their physiological function are unknown. We combine computational modeling, automated tracking of CCP dynamics, electron microscopy, and functional rescue experiments to demonstrate that CCP growth and lifetime are closely correlated and mechanistically linked by the early-acting endocytic F-BAR protein FCHo2.

Viscoelastic voyages - Biophysical perspectives on cell intercalation during Drosophila gastrulation.

Developmental processes are driven by a combination of cytoplasmic, cortical, and surface-associated forces. However, teasing apart the contributions of these forces and how a viscoelastic cell responds has long been a key question in developmental biology. Recent advances in applying biophysical approaches to these questions is leading to a fundamentally new understanding of morphogenesis.

Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constriction.

During gastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a trafficking pathway initiated by Sbf is disrupted, contractile steps become reversible.

Vertex sliding drives intercalation by radial coupling of adhesion and actomyosin networks during germband extension.

Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cellcell , while the function of tricellular has remained unaddressed. Here, we identify a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation.

Planar polarized Rab35 functions as an oscillatory ratchet during cell intercalation in the Drosophila epithelium.

The coordination between membrane trafficking and actomyosin networks is essential to the regulation of cell and tissue shape. Here, we examine Rab protein distributions during Drosophila epithelial tissue remodeling and show that Rab35 is dynamically planar polarized. Rab35 compartments are enriched at contractile interfaces of intercalating cells and provide the first evidence of interfacial monopolarity.

Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2.

The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown.

Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster.

One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminate in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events are unclear.

Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively.

Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation.

The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that do not require fluorescence tagging and that automatically track and characterize three aspects of scattering in live cells: increase in cell motility, loss of cell-cell adhesion, and spatial dispersion of cells (the redistribution of cells during scattering). We used these tools to screen a library of drugs, and we identified several efficient inhibitors of scattering, which we classified as selective inhibitors of either motility or loss of cell-cell adhesion, or as nonselective inhibitors.

RAB-5 controls the cortical organization and dynamics of PAR proteins to maintain C. elegans early embryonic polarity.

In all organisms, cell polarity is fundamental for most aspects of cell physiology. In many species and cell types, it is controlled by the evolutionarily conserved PAR-3, PAR-6 and aPKC proteins, which are asymmetrically localized at the cell cortex where they define specific domains. While PAR proteins define the antero-posterior axis of the early C.

Hotspots organize clathrin-mediated endocytosis by efficient recruitment and retention of nucleating resources.

The formation of clathrin-coated pits (CCPs) at the plasma membrane has been reported to sometimes occur repeatedly at predefined sites. However, defining such CCP 'hotspots' structurally and mechanistically has been difficult due to the dynamic and heterogeneous nature of CCPs. Here, we explore the molecular requirements for hotspots using a global assay of CCP dynamics.

Measuring the hierarchy of molecular events during clathrin-mediated endocytosis.

A well-orchestrated hierarchy of molecular events is required for successful initiation and maturation of clathrin-coated pits (CCPs). Nevertheless, CCPs display a broad range of lifetimes. This dynamic heterogeneity could either reflect differences in the temporal hierarchy of molecular events, or similar CCP maturation processes with variable kinetics.

Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis.

Clathrin-mediated endocytosis of surface receptors and their bound ligands (i.e., cargo) is highly regulated, including by the cargo itself.

Global and local regulation of clathrin-coated pit dynamics detected on patterned substrates.

Live-cell imaging of individual clathrin-coated pit (CCP) dynamics has revealed a broad variation in their internalization kinetics, but the functional significance and mechanistic underpinnings of this heterogeneity remain unknown. One contributing factor may be the spatial variations in the underlying actin cortex. To test this, we cultured cells on fibronectin (Fn) micropatterned substrates to vary the cortical actin mechanics in a defined manner.

Endocytic accessory proteins are functionally distinguished by their differential effects on the maturation of clathrin-coated pits.

Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin.

Cargo and dynamin regulate clathrin-coated pit maturation.

Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-coated pits (CCPs). To identify intermediates in the formation of clathrin-coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories.

Robust single-particle tracking in live-cell time-lapse sequences.

Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories.