MBI Videos

Workshop 6: Transport in a cell

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    John Cooper
    I will discuss work from our lab and other labs on how dynein functions to position the mitotic spindle and the nucleus in budding yeast. Interactions of cytoplasmic microtubules with the cortex control spindle position, and dynein represents one of the major pathways by which spindle position is controlled. Dynein functions by an "offloading" mechanism in which dynein targeted to dynamic microtubule plus ends is transferred to the cortex, where it is anchored and activated for minus-end directed motor activity, which pulls the spindle into the neck between mother and bud. Dynein is a multi-subunit complex, and dynein requires the function of dynactin, another multisubunit complex. Other factors are important for dynein targeting, both to the plus end and to the cortex. Dynein appears to be regulated spatially and temporally, during the course of its action. Finally, loss of dynein function and failure to properly position the spindle leads to activation of a cell-cycle checkpoint, which delays the progression of the cell cycle until alternative mechanisms are able to move the spindle into the mother / bud neck. The mechanism of this checkpoint relies on feedback information from cytoplasmic microtubules, based on new laser-cutting experiments.
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    Sarah Rice
    Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, to L5, and to the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of STLC, an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of nucleotide state.

    Work done in collaboration with Adam G. Larson, Nariman Naber, Roger Cooke, and Edward Pate.
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    Sean Sun
    Myosin is a major molecular force generator in the cell. The essential features of myosin interaction with actin filaments are understood. In the cell, the interactions of myosin with F-actin, substrate adhesions and other actin-associated proteins are less clear. We will borrow ideas from mathematical models of skeletal muscle to develop simple models for integrin focal adhesions, actin cross-linking proteins and non-muscle myosin-II. When these components are combined, a dynamical picture of the actin network emerges. In this talk, we will focus on the role of cell-substrate stiffness on the actin dynamics. Possible implications for mechanical sensing by cells are discussed.
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    Kristen Verhey
    We probe the transport properties in protein solutions stable with respect to any, solid or liquid, phase separation as a step in the understanding of transport in the cytosol of live cells. We determine the mean squared displacement of probe particles in the time range 1 millisecond - 10 seconds in solutions of a model protein. The tested solutions exhibit significant elasticity at high frequencies, while at low frequencies, they are purely viscous. We attribute this viscoelasticity to a dense network of weakly-bound chains of protein molecules with characteristic lifetime of 10-100 ms. The found intrinsic viscoelasticity of protein solutions should be considered in biochemical kinetics models.
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    Debashish Chowdhury
    Polymerases and ribosome are molecular machines which perform three important biological functions. Like cytoskeletal motors, each of these moves along a track using chemical energy for performing mechanical work. Moreover, it decodes genetic information chemically-encoded in the sequence of the subunits of the track. Furthermore, it polymerizes a macromolecule (DNA, RNA or protein) using the required subunits in a sequence that is dictated by the sequence of subunits of a template which serves also as its track for translocation. Enormous progress has been made in the last decade in understanding the structure and dynamics of these molecular machines using a combination of X-ray crystallography, cryo-electron microscopy, single-molecule imaging and manipulation. In recent years, we have developed models of these machines capturing the key features of their structure and dynamics to gain a quantitative understanding of their operational mechanism. We have also investigated the traffic-like collective movement of ribosomes simultaneously on the same mRNA track (and similar traffic of RNA polymerases on a DNA). We have also suggested new experiments for testing our theoretical predictions on the stochastic translocation-and-pause kinetics of a single motor as well as on their collective spatio-temporal organization.
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    Alexei Kornyshev
    DNA chasing DNA: physics of homology recognition and the secret of perfect match

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