Biomedical subjects

Roy Wollman

Publications and source records attributed to Roy Wollman.

6 recordsLinked to original sources

PubMed2006

A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays.

BACKGROUND: Mitosis depends upon the cooperative action of multiple microtubule (MT)-based motors. Among these, a kinesin-5, KLP61F, and the kinesin-14, Ncd, are proposed to generate antagonistic-sliding forces that control the spacing of the spindle poles. We tested whether purified KLP61F homotetramers and Ncd homodimers can generate a force balance capable of maintaining a constant spindle length in Drosophila embryos. RESULTS: Using fluorescence microscopy and cryo-EM, we observed that purified full-length, motorless, and tailless KLP61F tetramers (containing a tetramerization domain) and Ncd dimers can all cross-link MTs into bundles in MgATP. In multiple-motor motility assays, KLP61F and Ncd drive plus-end and minus-end MT sliding at 0.04 and 0.1 microm/s, respectively, but the motility of either motor is decreased by increasing the mole fraction of the other. At the "balance point," the mean velocity was zero and MTs paused briefly and then oscillated, taking approximately 0.3 microm excursions at approximately 0.02 microm/s toward the MT plus end and then the minus end. CONCLUSIONS: The results, combined with quantitative analysis, suggest that these motors could act as mutual brakes to modulate the rate of pole-pole separation and could maintain a prometaphase spindle displaying small fluctuations in its steady-state length.

Adenosine Triphosphate↗

PubMed2006

Modeling mitosis.

The mitotic spindle is a fascinating protein machine that uses bipolar arrays of dynamic microtubules and many mitotic motors to coordinate the accurate segregation of sister chromatids. Here we discuss recent mathematical models and computer simulations that, in concert with experimental studies, help explain the molecular mechanisms by which the spindle machinery performs its crucial functions. We review current models of spindle assembly, positioning, maintenance and elongation; of chromosome capture and congression; and of the spindle assembly checkpoint. We discuss some limitations of the application of modeling to other aspects of mitosis and the feasibility of building more comprehensive system-level models.

Animals↗

PubMed2006

Quantitative modeling in cell biology: what is it good for?

Recently, there has been a surge in the number of pioneering studies combining experiments with quantitative modeling to explain both relatively simple modules of molecular machinery of the cell and to achieve system-level understanding of cellular networks. Here we discuss the utility and methods of modeling and review several current models of cell signaling, cytoskeletal self-organization, nuclear transport, and the cell cycle. We discuss successes of and barriers to modeling in cell biology and its future directions, and we argue, using the field of bacterial chemotaxis as an example, that the closer the complete systematic understanding of cell behavior is, the more important modeling becomes and the more experiment and theory merge.

Active Transport, Cell Nucleus↗

PubMed2005

Length control of the metaphase spindle.

BACKGROUND: The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. RESULTS: Using the Drosophila S2 cell line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. CONCLUSIONS: By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.

Animals↗

PubMed2005

APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment.

Recently, we have shown that a cancer causing truncation in adenomatous polyposis coli (APC) (APC(1-1450)) dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. Here, we examine the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. We show that siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. We show that APC(1-1450) acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, which is consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC(1-1450) compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2005

QuasiMotiFinder: protein annotation by searching for evolutionarily conserved motif-like patterns.

Sequence signature databases such as PROSITE, which include amino acid segments that are indicative of a protein's function, are useful for protein annotation. Lamentably, the annotation is not always accurate. A signature may be falsely detected in a protein that does not carry out the associated function (false positive prediction, FP) or may be overlooked in a protein that does carry out the function (false negative prediction, FN). A new approach has emerged in which a signature is replaced with a sequence profile, calculated based on multiple sequence alignment (MSA) of homologous proteins that share the same function. This approach, which is superior to the simple pattern search, essentially searches with the sequence of the query protein against an MSA library. We suggest here an alternative approach, implemented in the QuasiMotiFinder web server (http://quasimotifinder.tau.ac.il/), which is based on a search with an MSA of homologous query proteins against the original PROSITE signatures. The explicit use of the average evolutionary conservation of the signature in the query proteins significantly reduces the rate of FP prediction compared with the simple pattern search. QuasiMotiFinder also has a reduced rate of FN prediction compared with simple pattern searches, since the traditional search for precise signatures has been replaced by a permissive search for signature-like patterns that are physicochemically similar to known signatures. Overall, QuasiMotiFinder and the profile search are comparable to each other in terms of performance. They are also complementary to each other in that signatures that are falsely detected in (or overlooked by) one may be correctly detected by the other.

Amino Acid Motifs↗