Search

Accurate chromosome segregation depends on proper kinetochore-microtubule attachment. Upon microtubule interaction, kinetochores are subjected to forces generated by the microtubules. In this work, we used laser ablation to sever microtubules attached to a merotelic kinetochore, which is laterally stretched by opposing pulling forces exerted by microtubules, and inferred the mechanical response of the kinetochore from its length change. In both mammalian PtK1 cells and in the fission yeast Schizosaccharomyces pombe, kinetochores shortened after microtubule severing. Interestingly, the inner kinetochore-centromere relaxed faster than the outer kinetochore. Whereas in fission yeast all kinetochores relaxed to a similar length, in PtK1 cells the more stretched kinetochores remained more stretched. Simple models suggest that these differences arise because the mechanical structure of the mammalian kinetochore is more complex. Our study establishes merotelic kinetochores as an experimental model for studying the mechanical response of the kinetochore in live cells and reveals a viscoelastic behavior of the kinetochore that is conserved in yeast and mammalian cells.

Chromosome ends contain nucleoprotein structures known as telomeres. Damage to chromosome ends during interphase elicits a DNA damage response (DDR) resulting in cell cycle arrest. However, little is known regarding the signaling from damaged chromosome ends (designated here as "TIPs") during mitosis. In the present study, we investigated the consequences of DNA damage induced at a single TIP in mitosis. We used laser microirradiation to damage mitotic TIPs or chromosome arms (non-TIPs) in PtK2 kidney epithelial cells. We found that damage to a single TIP, but not a non-TIP, delays anaphase onset. This TIP-specific checkpoint response is accompanied by differential recruitment of DDR proteins. Although phosphorylation of H2AX and the recruitment of several repair factors, such as Ku70-Ku80, occur in a comparable manner at both TIP and non-TIP damage sites, DDR factors such as ataxia telangiectasia mutated (ATM), MDC1, WRN, and FANCD2 are specifically recruited to TIPs but not to non-TIPs. In addition, Nbs1, BRCA1, and ubiquitin accumulate at damaged TIPs more rapidly than at damaged non-TIPs. ATR and 53BP1 are not detected at either TIPs or non-TIPs in mitosis. The observed delay in anaphase onset is dependent on the activity of DDR kinases ATM and Chk1, and the spindle assembly checkpoint kinase Mps1. Cells damaged at a single TIP or non-TIP eventually exit mitosis with unrepaired lesions. Damaged TIPs are segregated into micronuclei at a significantly higher frequency than damaged non-TIPs. Together, these findings reveal a mitosis-specific DDR uniquely associated with chromosome ends.

In this chapter, we review the imaging techniques and methods of molecular interrogation made possible by integrating laser light sources with microscopy. We discuss the advantages of exciting fluorescence by laser illumination and review commonly used laser-based imaging techniques such as confocal, multiphoton, and total internal reflection microcopy. We also discuss emerging imaging modalities based on intrinsic properties of biological macromolecules such as second harmonic generation imaging and coherent anti-Raman resonance spectroscopy. Super resolution techniques are presented that exceed the theoretical diffraction-limited resolution of a microscope objective. This chapter also focuses on laser-based techniques that can report biophysical parameters of fluorescently labeled molecules within living cells. Photobleaching techniques, fluorescence lifetime imaging, and fluorescence correlation methods can measure kinetic rates, molecular diffusion, protein-protein interactions, and concentration of a fluorophore-bound molecule. This chapter provides an introduction to the field of laser-based microscopy enabling readers to determine how best to match their research questions to the current suite of techniques.

BackgroundThe mitotic checkpoint prevents the onset of anaphase before all chromosomes are attached to spindle microtubules. The checkpoint is thought to act by the catalytic generation at unattached kinetochores of a diffusible "wait signal" that prevents anaphase. Mad2 and Cdc20, two candidate proteins for components of a diffusible wait signal, have previously been shown to be recruited to and rapidly released from unattached kinetochores.ResultsFluorescence recovery after photobleaching demonstrated that Mad1, Bub1, and a portion of Mad2, all essential mitotic-checkpoint components, are stably bound elements of unattached kinetochores (as are structural centromere components such as Centromere protein C [CENP-C]). After microtubule attachment, Mad1 and Mad2 are released from kinetochores and relocalize to spindle poles, whereas Bub1 remains at kinetochores.ConclusionsA long residence time at kinetochores identifies Bub1, Mad1, and a portion of Mad2 as part of a catalytic platform that recruits, activates, and releases a diffusible wait signal that is partly composed of the rapidly exchanging portion of Mad2. The release of Mad1 and Mad2, but not Bub1, from kinetochores upon attachment separates the elements of this "catalytic platform" and thereby silences generation of the anaphase inhibitor despite continued rapid cycling of Mad2 at spindle poles.

Fibrosis is responsible for chronic progressive kidney failure, which is present in a large number of adults in the developed world. It is increasingly appreciated that acute kidney injury (AKI), resulting in aberrant incomplete repair, is a major contributor to chronic fibrotic kidney disease. The mechanism that triggers the fibrogenic response after injury is not well understood. In ischemic, toxic and obstructive models of AKI, we demonstrate a causal association between epithelial cell cycle G2/M arrest and a fibrotic outcome. G2/M-arrested proximal tubular cells activate c-jun N[H.sub.2]-terminal kinase (JNK) signaling, which acts to upregulate profibrotic cytokine production. Treatment with a JNK inhibitor, or bypassing the G2/M arrest by administration of a p53 inhibitor or the removal of the contralateral kidney, rescues fibrosis in the unilateral ischemic injured kidney. Hence, epithelial cell cycle arrest at G2/M and its subsequent downstream signaling are hitherto unrecognized therapeutic targets for the prevention of fibrosis and interruption of the accelerated progression of kidney disease., The repair process after tissue injury involves a regeneration phase, in which injured cells are replaced by cells of the same lineage (1), and a fibrotic phase, in which connective [...]

The review discusses regulation of the cell cycle events with particular reference to anaphase stage of cell division. Data point out that Mad2 protein possessing ubiquitination and proteolytic machinery participates in the signal transmission for the spindle checkpoint.

Vimentin intermediate filaments are a major cytoskeletal constituent of cells of mesenchymal origin. They have been colocalized with a variety of intracellular structures such as actin filaments and the plasma [...]

The three-dimensional polymer network formed by the cytoskeleton is the main determinant of cellular mechanics (Elson, 1988; Maniotis et al., 1997) and is required for the cell to resist external [...]