Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus.

Research in cellular mechanotransduction often focuses on how extracellular physical forces are converted into chemical signals at the cell surface. However, mechanical forces that are exerted on surface-adhesion receptors, such as integrins and cadherins, are also channelled along cytoskeletal filaments and concentrated at distant sites in the cytoplasm and nucleus. Here, we explore the molecular mechanisms by which forces might act at a distance to induce mechanochemical conversion in the nucleus and alter gene activities.

Analysis of kinesin motor function at budding yeast kinetochores.

Accurate chromosome segregation during mitosis requires biorientation of sister chromatids on the microtubules (MT) of the mitotic spindle. Chromosome-MT binding is mediated by kinetochores, which are multiprotein structures that assemble on centromeric (CEN) DNA. The simple CENs of budding yeast are among the best understood, but the roles of kinesin motor proteins at yeast kinetochores have yet to be determined, despite evidence of their importance in higher eukaryotes. We show that all four nuclear kinesins in Saccharomyces cerevisiae localize to kinetochores and function in three distinct processes. Kip1p and Cin8p, which are kinesin-5/BimC family members, cluster kinetochores into their characteristic bilobed metaphase configuration. Kip3p, a kinesin-8,-13/KinI kinesin, synchronizes poleward kinetochore movement during anaphase A. The kinesin-14 motor Kar3p appears to function at the subset of kinetochores that become detached from spindle MTs. These data demonstrate roles for structurally diverse motors in the complex processes of chromosome segregation and reveal important similarities and intriguing differences between higher and lower eukaryotes.

Mechanical control of cAMP signaling through integrins is mediated by the heterotrimeric Galphas protein.

Mechanical stresses that are preferentially transmitted across the cell surface via transmembrane integrin receptors activate gene transcription by triggering production of intracellular chemical second messengers, such as cAMP. Here we show that the sensitivity of the cAMP signaling pathway to mechanical stresses transferred across beta1 integrins is mediated by force-dependent activation of the heterotrimeric G protein subunit Galphas within focal adhesions at the site of stress application. Galphas is recruited to focal adhesions that form within minutes following clustering of beta1 integrins induced by cell binding to magnetic microbeads coated with activating integrin ligands, and beta1 integrin and Galphas co-precipitate when analyzed biochemically. Stress application to activated beta1 integrins using magnetic twisting cytometry increases Galphas recruitment and activates these large G proteins within focal adhesions, as measured by binding of biotinylated azido-anilido-GTP, whereas application of similar stresses to inactivated integrins or control histocompatibility antigens has little effect. This response is relevant physiologically as application of mechanical strain to cells bound to flexible extracellular matrix-coated substrates induce translocation of phospho-CREB to the nucleus, which can be attenuated by inhibiting Galphas activity, either using the inhibitor melittin or suppressing its expression using siRNA. Although integrins are not typical G protein-coupled receptors, these results show that integrins focus mechanical stresses locally on heterotrimeric G proteins within focal adhesions at the site of force application, and transduce mechanical stimuli into an intracellular cAMP signaling response by activating Galphas at these membrane signaling sites.

Functional hierarchy of redundant actin assembly factors revealed by fine-grained registration of intrinsic image fluctuations.

Highly redundant pathways often contain components whose functions are difficult to decipher from the responses induced by genetic or molecular perturbations. Here, we present a statistical approach that samples and registers events observed in images of intrinsic fluctuations in unperturbed cells to establish the functional hierarchy of events in systems with redundant pathways. We apply this approach to study the recruitment of actin assembly factors involved in the protrusion of the cell membrane. We find that the formin mDia1, along with nascent adhesion components, is recruited to the leading edge of the cell before protrusion onset, initiating linear growth of the lamellipodial network. Recruitment of Arp2/3, VASP, cofilin, and the formin mDia2 then promotes sustained exponential growth of the network. Experiments changing membrane tension suggest that Arp2/3 recruitment is mechano-responsive. These results indicate that cells adjust the overlapping contributions of multiple factors to actin filament assembly during protrusion on a ten-second timescale and in response to mechanical cues.

Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface beta1 integrins.

Integrins are ubiquitous transmembrane mechanoreceptors that elicit changes in intracellular biochemistry in response to mechanical force application, but these alterations generally proceed over seconds to minutes. Stress-sensitive ion channels represent another class of mechanoreceptors that are activated much more rapidly (within msec), and recent findings suggest that calcium influx through Transient Receptor Potential Vanilloid-4 (TRPV4) channels expressed in the plasma membrane of bovine capillary endothelial cells is required for mechanical strain-induced changes in focal adhesion assembly, cell orientation and directional migration. However, whether mechanically stretching a cell's extracellular matrix (ECM) adhesions might directly activate cell surface ion channels remains unknown. Here we show that forces applied to beta1 integrins result in ultra-rapid (within 4 msec) activation of calcium influx through TRPV4 channels. The TRPV4 channels were specifically activated by mechanical strain in the cytoskeletal backbone of the focal adhesion, and not by deformation of the lipid bilayer or submembranous cortical cytoskeleton alone. This early-immediate calcium signaling response required the distal region of the beta1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and external force application to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response. Local direct strain-dependent activation of TRPV4 channels mediated by force transfer from integrins and CD98 may therefore enable compartmentalization of calcium signaling within focal adhesions that is critical for mechanical control of many cell behaviors that underlie cell and tissue development.

Comparative autoregressive moving average analysis of kinetochore microtubule dynamics in yeast.

To elucidate the regulation of kinetochore microtubules (kMTs) by kinetochore proteins in Saccharomyces cerevisiae, we need tools to characterize and compare stochastic kMT dynamics. Here we show that autoregressive moving average (ARMA) models, combined with a statistical framework for testing the significance of differences between ARMA model parameters, provide a sensitive method for identifying the subtle changes in kMT dynamics associated with kinetochore protein mutations. Applying ARMA analysis to G1 kMT dynamics, we found that 1), kMT dynamics in the kinetochore protein mutants okp1-5 and kip3Delta are different from those in wild-type, demonstrating the regulation of kMTs by kinetochore proteins; 2), the kinase Ipl1p regulates kMT dynamics also in G1; and 3), the mutant dam1-1 exhibits three different phenotypes, indicating the central role of Dam1p in maintaining the attachment of kMTs and regulating their dynamics. We also confirmed that kMT dynamics vary with temperature, and are most likely differentially regulated at 37 degrees C. Therefore, when elucidating the role of a protein in kMT regulation using a temperature-sensitive mutant, dynamics in the mutant at its nonpermissive temperature must be compared to those in wild-type at the same temperature, not to those in the mutant at its permissive temperature.

Structure, function, and regulation of budding yeast kinetochores.

Kinetochores are multiprotein complexes that assemble on centromeric DNA and mediate the attachment and movement of chromosomes along the microtubules (MTs) of the mitotic spindle. This review focuses on the simplest eukaryotic centromeres and kinetochores, those found in the budding yeast Saccharomyces cerevisiae. Research on kinetochore function and chromosome segregation is focused on four questions of general significance: what specifies the location of centromeres? What are the protein components of kinetochores, and how do they assemble a MT attachment site? How do MT attachments generate force? How do cells sense the state of attachment via the spindle assembly checkpoint?