Evolution of cellular morpho-phenotypes in cancer metastasis.

Intratumoral heterogeneity greatly complicates the study of molecular mechanisms driving cancer progression and our ability to predict patient outcomes. Here we have developed an automated high-throughput cell-imaging platform (htCIP) that allows us to extract high-content information about individual cells, including cell morphology, molecular content and local cell density at single-cell resolution. We further develop a comprehensive visually-aided morpho-phenotyping recognition (VAMPIRE) tool to analyze irregular cellular and nuclear shapes in both 2D and 3D microenvironments.

The LINC-anchored actin cap connects the extracellular milieu to the nucleus for ultrafast mechanotransduction.

Cells continuously sense and respond to external mechanical forces through their cytoskeleton. Here we show that only a small subset of actin fibers, those forming the perinuclear actin cap that wraps around the nucleus, form in response to low physiological mechanical stresses in adherent fibroblasts. While conventional basal stress fibers form only past a threshold shear stress of 0.

Functional interplay between the cell cycle and cell phenotypes.

Cell cycle distribution of adherent cells is typically assessed using flow cytometry, which precludes the measurements of many cell properties and their cycle phase in the same environment. Here we develop and validate a microscopy system to quantitatively analyze the cell-cycle phase of thousands of adherent cells and their associated cell properties simultaneously. This assay demonstrates that population-averaged cell phenotypes can be written as a linear combination of cell-cycle fractions and phase-dependent phenotypes.

Actin cap associated focal adhesions and their distinct role in cellular mechanosensing.

The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to biochemical stimuli. Actin cap associated focal adhesions (ACAFAs) dominate cell mechanosensing over a wide range of matrix stiffness, an ACAFA-specific function regulated by actomyosin contractility in the actin cap, while conventional focal adhesions are restrictively involved in mechanosensing for extremely soft substrates.

The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration.

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles - the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration.

The differential formation of the LINC-mediated perinuclear actin cap in pluripotent and somatic cells.

The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation.

SMRT analysis of MTOC and nuclear positioning reveals the role of EB1 and LIC1 in single-cell polarization.

In several migratory cells, the microtubule-organizing center (MTOC) is repositioned between the leading edge and nucleus, creating a polarized morphology. Although our understanding of polarization has progressed as a result of various scratch-wound and cell migration studies, variations in culture conditions required for such assays have prevented a unified understanding of the intricacies of MTOC and nucleus positioning that result in cell polarization. Here, we employ a new SMRT (for sparse, monolayer, round, triangular) analysis that uses a universal coordinate system based on cell centroid to examine the pathways regulating MTOC and nuclear positions in cells plated in a variety of conditions.

The perinuclear actin cap in health and disease.

We recently demonstrated the existence of a previously uncharacterized subset of actomyosin fibers that form the perinuclear actin cap, a cytoskeletal structure that tightly wraps around the nucleus of a wide range of somatic cells. Fibers in the actin cap are distinct from well-characterized, conventional actin fibers at the basal and dorsal surfaces of adherent cells in their subcellular location, internal organization, dynamics, ability to generate contractile forces, response to cytoskeletal pharmacological treatments, response to biochemical stimuli, regulation by components of the linkers of nucleoskeleton and cytoskeleton (LINC) complexes, and response to disease-associated mutations in LMNA, the gene that encodes for the nuclear lamin component lamin A/C. The perinuclear actin cap precisely shapes the nucleus in interphase cells.

Differences in the microrheology of human embryonic stem cells and human induced pluripotent stem cells.

Embryonic and adult fibroblasts can be returned to pluripotency by the expression of reprogramming genes. Multiple lines of evidence suggest that these human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells are behaviorally, karyotypically, and morphologically similar. Here we sought to determine whether the physical properties of hiPS cells, including their micromechanical properties, are different from those of hES cells.

A perinuclear actin cap regulates nuclear shape.

Defects in nuclear morphology often correlate with the onset of disease, including cancer, progeria, cardiomyopathy, and muscular dystrophy. However, the mechanism by which a cell controls its nuclear shape is unknown. Here, we use adhesive micropatterned surfaces to control the overall shape of fibroblasts and find that the shape of the nucleus is tightly regulated by the underlying cell adhesion geometry.

Dysfunctional connections between the nucleus and the actin and microtubule networks in laminopathic models.

Laminopathies encompass a wide array of human diseases associated to scattered mutations along LMNA, a single gene encoding A-type lamins. How such genetic alterations translate to cellular defects and generate such diverse disease phenotypes remains enigmatic. Recent work has identified nuclear envelope proteins--emerin and the linker of the nucleoskeleton and cytoskeleton (LINC) complex--which connect the nuclear lamina to the cytoskeleton.

Nuclear lamin A/C deficiency induces defects in cell mechanics, polarization, and migration.

Lamin A/C is a major constituent of the nuclear lamina, a thin filamentous protein layer that lies beneath the nuclear envelope. Here we show that lamin A/C deficiency in mouse embryonic fibroblasts (Lmna(-/-) MEFs) diminishes the ability of these cells to polarize at the edge of a wound and significantly reduces cell migration speed into the wound. Moreover, lamin A/C deficiency induces significant separation of the microtubule organizing center (MTOC) from the nuclear envelope.

Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow.

Cells in vivo are constantly subjected to mechanical shear stresses that play important regulatory roles in various physiological and pathological processes. Cytoskeletal reorganizations that occur in response to shear flow have been studied extensively, but whether the cytoplasm of an adherent cell adapts its mechanical properties to respond to shear is largely unknown. Here we develop a new method where fluorescent nanoparticles are ballistically injected into the cells to probe, with high resolution, possible local viscoelastic changes in the cytoplasm of individual cells subjected to fluid flow.