Genetic analysis reveals that FLO11 upregulation and cell polarization independently regulate invasive growth in Saccharomyces cerevisiae.

Under inducing conditions, haploid Saccharomyces cerevisiae perform a dimorphic transition from yeast-form growth on the agar surface to invasive growth, where chains of cells dig into the solid growth medium. Previous work on signaling cascades that promote agar invasion has demonstrated upregulation of FLO11, a cell-surface flocculin involved in cell-cell adhesion. We find that increasing FLO11 transcription is sufficient to induce both invasive and filamentous growth. A genetic screen for repressors of FLO11 isolated mutant strains that dig into agar (dia) and identified mutations in 35 different genes: ELM1, HSL1, HSL7, BUD3, BUD4, BUD10, AXL1, SIR2, SIR4, BEM2, PGI1, GND1, YDJ1, ARO7, GRR1, CDC53, HSC82, ZUO1, ADH1, CSE2, GCR1, IRA1, MSN5, SRB8, SSN3, SSN8, BPL1, GTR1, MED1, SKN7, TAF25, DIA1, DIA2, DIA3, and DIA4. Indeed, agar invasion in 20 dia mutants requires upregulation of the endogenous FLO11 promoter. However, 13 mutants promote agar invasion even with FLO11 clamped at a constitutive low-expression level. These FLO11 promoter-independent dia mutants establish distinct invasive growth pathways due to polarized bud site selection and/or cell elongation. Epistasis with the STE MAP kinase cascade and cytokinesis/budding checkpoint shows these pathways are targets of DIA genes that repress agar invasion by FLO11 promoter-dependent and -independent mechanisms, respectively.

Intermolecular electrostatic interactions and their effect on flux and protein deposition during protein filtration.

Although membrane filtration is used extensively to process protein solutions containing a variety of electrolytes, there is currently little fundamental understanding of the effect of the solution environment (and in particular, the solution pH) on the filtrate flux in these systems. We have obtained data for the flux and sieving coefficients during the batch (stirred cell) filtration of solutions of bovine serum albumin, immunoglobulins, hemoglobin, ribonuclease A, and lysozyme through 0.16-micron microfiltration membranes at different pH values. The flux declined significantly for all five proteins due to the formation of a protein deposit on the upper surface of the membrane. The quasi-steady ultrafiltrate fluxes at the individual protein isoelectric pH's were essentially identical, despite the large differences in molecular weight and physicochemical characteristics of these proteins. The flux increased at pH's away from the isoelectric point, with the data well-correlated with the protein surface charge density. These results were explained in terms of a simple physical model in which the protein deposit continues to grow, and thus the flux continues to decline, until the drag force on the proteins associated with the filtrate flow is no longer able to overcome the intermolecular repulsive interactions between the proteins in the bulk solution and those in the protein deposit on the surface of the membrane.

Engineering tissue from human embryonic stem cells.

Recent advances in human embryonic stem cell (hESC) biology now offer an alternative cell source for tissue engineers, as these cells are capable of proliferating indefinitely and differentiating to many clinically relevant cell types. Novel culture methods capable of exerting spatial and temporal control over the stem cell microenvironment allow for more efficient expansion of hESCs, and significant advances have been made toward improving our understanding of the biophysical and biochemical cues that direct stem cell fate choices. Effective production of lineage specific progenitors or terminally differentiated cells enables researchers to incorporate hESC derivatives into engineered tissue constructs. Here, we describe current efforts using hESCs as a cell source for tissue engineering applications, highlighting potential advantages of hESCs over current practices as well as challenges which must be overcome.

Kinetic model for integrin-mediated adhesion release during cell migration.

Under many circumstances, cell migration speed is limited by the rate of cell-substratum detachment at the cell rear. We have constructed a mathematical model to integrate how the biophysical and biochemical interactions between integrins, the cytoskeleton, and the matrix affect rear retraction and linkage dissociation mechanisms. Our model also examines how applied forces and integrin clustering affect retraction kinetics. The model predicts two distinct detachment phenotypes. In the first, detachment is extremely rapid, dominated by integrin extracellular-matrix dissociation, and it occurs at high forces or low adhesiveness. In the second, detachment is much slower, dominated by integrin-cytoskeleton dissociation, and it occurs at low forces or high adhesiveness. The amount of integrin extracted from the rear of the cell is an assay for the detachment phenotype. During rapid detachment cells leave little integrin on the substratum whereas during slow detachment a large fraction of integrin rips from the membrane. This model delineates parameters which can be exploited to regulate cell speed in each detachment regime. The model also offers an explanation as to why some cell types, such as leukocytes or keratocytes, are able to detach easily and move very quickly while other cell types, such as fibroblasts, tend to migrate more slowly and release many more integrins during detachment.

Physical and biochemical regulation of integrin release during rear detachment of migrating cells.

Cell migration can be considered as a repeated cycle of membrane protrusion and attachment, cytoskeletal contraction and rear detachment. At intermediate and high levels of cell-substratum adhesiveness, cell speed appears to be rate-limited by rear detachment, specifically by the disruption of cytoskeleton-adhesion receptor-extracellular matrix (ECM) linkages. Often, cytoskeletal linkages fracture to release integrin adhesion receptors from the cell. Cell-extracellular matrix bonds may also dissociate, allowing the integrins to remain with the cell. To investigate molecular mechanisms involved in fracturing these linkages and regulating cell speed, we have developed an experimental system to track integrins during the process of rear retraction in Chinese hamster ovary (CHO) cells. Integrin expression level was varied by transfecting CHO B2 cells, which express very little endogenous alpha5 integrin, with a plasmid containing human alpha5 integrin cDNA and sorting the cells into three populations with different alpha5 expression levels. Receptor/ligand affinity was varied using CHO cells transfected with either alphaIIbbeta3 or alphaIIbbeta3(beta1-2), a high affinity variant. alphaIIbbeta3(beta1-2) is activated to a higher affinity state with an anti-LIBS2 antibody. Fluorescent probes were conjugated to non-adhesion perturbing anti-integrin antibodies, which label integrins in CHO cells migrating on a matrix-coated glass coverslip. The rear retraction area was determined using phase contrast microscopy and integrins initially in this area were tracked by fluorescence microscopy and a cooled CCD camera. We find that rear retraction rate appears to limit cell speed at intermediate and high adhesiveness, but not at low adhesiveness. Upon rear retraction, the amount of integrin released from the cell increases as extracellular matrix concentration, receptor level and receptor-ligand affinity increase. In fact, integrin release is a constant function of cell-substratum adhesiveness and the number of cell-substratum bonds. In the adhesive regime where rear detachment limits the rate of cell migration, cell speed has an inverse relationship to the amount of integrin released at the rear of the cell. At high cell-substratum adhesiveness, calpain, a Ca2+-dependent protease, is also involved in release of cytoskeletal linkages during rear retraction. Inhibition of calpain results in decreased integrin release from the cell membrane, and consequently a decrease in cell speed, during migration. These observations suggest a model for rear retraction in which applied tension and calpain-mediated cytoskeletal linkage cleavage are required at high adhesiveness, but only applied tension is required at low adhesiveness.

Integrin dynamics on the tail region of migrating fibroblasts.

Cell migration is a complex process that can be considered as a repeated cycle of lamellipod extension and attachment, cytoskeletal contraction, and tail detachment. While lamellipodial and cytoskeletal phenomena are currently the focus of considerable research on cell migration, under many conditions locomotion appears to be rate-limited by events at the cell rear, especially release of cell/substratum adhesions. To study the mechanism of tail detachment, we have developed a novel experimental system that permits observation of integrin dynamics on the ventral surface of migrating fibroblasts. Photoactivatable caged fluorescein is coupled to a non-adhesion-perturbing anti-avian-beta 1 integrin subunit antibody, which labels integrins on chicken fibroblasts migrating on a laminin-coated glass coverslip. Ultraviolet light is focused through a pinhole to photoactivate the caged fluorophore in a 10-micron-diameter spot at the rear of a polarized cell. The fate of integrins initially present in this spot is monitored using a cooled CCD camera to follow the movement of fluorescent intensity as a function of time over a 2 to 3 hour period. We find that a substantial fraction of the integrins is left behind on the substratum as the cell detaches and locomotes, while another fraction collects into vesicles which are transported along the cell body as the cell migrates. As aggregates rip from the cell membrane, the integrin-cytoskeletal bonds are preferentially fractured resulting in 81 +/- 15% of the integrin remaining attached to the substratum. We additionally find that adhesions sometimes disperse into integrins which can form new adhesions at other locations in the cell. Adhesions along the cell edge can release from the substrate and translocate with the cell. They either disperse in the cell membrane, rip from the cell membrane and remain attached to the substratum, or form a new aggregate. These observations indicate that the behavior of integrins at the cell rear is much more dynamic than previously appreciated, suggesting that an important locus for regulation of motility may reside in this region.

Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness.

Migration of cells in higher organisms is mediated by adhesion receptors, such as integrins, that link the cell to extracellular-matrix ligands, transmitting forces and signals necessary for locomotion. Whether cells will migrate or not on a given substratum, and also their speed, depends on several variables related to integrin-ligand interactions, including ligand levels, integrin levels, and integrin-ligand binding affinities. These and other factors affect the way molecular systems integrate to effect and regulate cell migration. Here we show that changes in cell migration speed resulting from three separate variables-substratum ligand level, cell integrin expression level, and integrin-ligand binding affinity-are all quantitatively predictable through the changes they cause in a single unifying parameter: short-term cell-substratum adhesion strength. This finding is consistent with predictions of a mathematical model for cell migration. The ligand concentration promoting maximum migration speed decreases reciprocally as integrin expression increases. Increases in integrin-ligand affinity similarly result in maximal migration at reciprocally lower ligand concentrations. The maximum speed attainable, however, remains unchanged as ligand concentration, integrin expression, or integrin-ligand affinity vary, suggesting that integrin coupling with intracellular motors remains unaltered.

Regulation of cell migration by the calcium-dependent protease calpain.

Integrin receptors play an important role during cell migration by mediating linkages and transmitting forces between the extracellular matrix and the actin cytoskeleton. The mechanisms by which these linkages are regulated and released during migration are not well understood. We show here that cell-permeable inhibitors of the calcium-dependent protease calpain inhibit both beta1 and beta3 integrin-mediated cell migration. Calpain inhibition specifically stabilizes peripheral focal adhesions, increases adhesiveness, and decreases the rate of cell detachment. Furthermore, these inhibitors alter the fate of integrin receptors at the rear of the cell during migration. A Chinese hamster ovary cell line expressing low levels of calpain I also shows reduced migration rates with similar morphological changes, further implicating calpain in this process. Taken together, the data suggest that calpain inhibition modulates cell migration by stabilizing cytoskeletal linkages and decreasing the rate of retraction of the cell's rear. Inhibiting calpain-mediated proteolysis may therefore be a potential therapeutic approach to control pathological cell migration such as tumor metastasis.