Cell-Length-Dependent Microtubule Accumulation during Polarization

SummaryBackgroundBreaking cell symmetry, known as polarization, requires dynamic reorganization of microtubules (MTs) and is essential to many cellular processes, including axon formation in neurons. A critical step in polarization is believed to be the “selective stabilization” of MTs, which hypothesizes a spatial and/or temporal shift toward net MT assembly in a preferred direction of growth.ResultsWe now find that a simpler “length-dependent” model, in which MT assembly parameters are spatially and temporally constant, predicts MT accumulation in the direction of growth because of longer mean first passage times in the longer direction. We experimentally tested both models by tracking MT assembly dynamics in polarizing embryonic chick forebrain neurons, and we confirmed that assembly is spatially and temporally constant during axon formation.ConclusionCell polarization occurs most simply through cell-length-dependent accumulation of MTs without MT stabilization or capture. In this way, F-actin-mediated cell shape and size changes can be read out by dynamic MTs undergoing simple dynamic instability to ultimately break cell symmetry

Optical Control of Microtubule Dynamics in Time and Space

Small molecule inhibitors of microtubule dynamics are widely used as cell biology research tools and clinically as cancer chemotherapeutics. By slight modification to the chemical structure of a known microtubule inhibitor, combretastatin A-4, Borowiak et al. develop a photoswitchable derivative that can be turned “on” and “off” with low-intensity light to spatially and temporally control microtubule dynamics

Regulation of the MEX-5 Gradient by a Spatially Segregated Kinase/Phosphatase Cycle

SummaryProtein concentration gradients encode spatial information across cells and tissues and often depend on spatially localized protein synthesis. Here, we report that a different mechanism underlies the MEX-5 gradient. MEX-5 is an RNA-binding protein that becomes distributed in a cytoplasmic gradient along the anterior-to-posterior axis of the one-cell C. elegans embryo. We demonstrate that the MEX-5 gradient is a direct consequence of an underlying gradient in MEX-5 diffusivity. The MEX-5 diffusion gradient arises when the PAR-1 kinase stimulates the release of MEX-5 from slow-diffusive, RNA-containing complexes in the posterior cytoplasm. PAR-1 directly phosphorylates MEX-5 and is antagonized by the spatially uniform phosphatase PP2A. Mathematical modeling and in vivo observations demonstrate that spatially segregated phosphorylation and dephosphorylation reactions are sufficient to generate stable protein concentration gradients in the cytoplasm. The principles demonstrated here apply to any spatially segregated modification cycle that affects protein diffusion and do not require protein synthesis or degradation

Stable Kinetochore-Microtubule Attachment Constrains Centromere Positioning in Metaphase

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle

Kinesin-8 molecular motors: putting the brakes on chromosome oscillations

Recent studies suggest that the human Kinesin-8 molecular motor Kif18A has a role in chromosome congression. Specifically, these studies find that Kif18A promotes chromosome congression by attenuating chromosome oscillation magnitudes. Together with recent modeling work, in vitro studies, and the analysis of in vivo yeast data, these reports reveal how Kinesin-8 molecular motors might control chromosome oscillation amplitudes by spatially regulating the dynamic instability of microtubule plus-ends within the mitotic spindle

RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage–induced cell senescence

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 27 (2016): 1346-1357, doi:10.1091/mbc.E16-01-0025.The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase–regulated nuclear–cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage–induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β–dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP–regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage.P.C., K.H., Y.P., J.-Q.C., M.A.H., S.K., and P.K. were supported by the Intramural Research Program of the Center for Cancer Research, NCI. D.O. and E.T. were supported by National Institutes of Health grant R01 GM071522

The microtubule-based motor Kar3 and plus end–binding protein Bim1 provide structural support for the anaphase spindle

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ∼8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome