Alan J. Hunt

Centrosome misorientation reduces stem cell division during ageing.

Asymmetric division of adult stem cells generates one self-renewing stem cell and one differentiating cell, thereby maintaining tissue homeostasis. A decline in stem cell function has been proposed to contribute to tissue ageing, although the underlying mechanism is poorly understood. Here we show that changes in the stem cell orientation with respect to the niche during ageing contribute to the decline in spermatogenesis in the male germ line of Drosophila. Throughout the cell cycle, centrosomes in germline stem cells (GSCs) are oriented within their niche and this ensures asymmetric division. We found that GSCs containing misoriented centrosomes accumulate with age and that these GSCs are arrested or delayed in the cell cycle. The cell cycle arrest is transient, and GSCs appear to re-enter the cell cycle on correction of centrosome orientation. On the basis of these findings, we propose that cell cycle arrest associated with centrosome misorientation functions as a mechanism to ensure asymmetric stem cell division, and that the inability of stem cells to maintain correct orientation during ageing contributes to the decline in spermatogenesis. We also show that some of the misoriented GSCs probably originate from dedifferentiation of spermatogonia.

MICROTUBULE ASSEMBLY DYNAMICS AT THE NANOSCALE

BACKGROUND The labile nature of microtubules is critical for establishing cellular morphology and motility, yet the molecular basis of assembly remains unclear. Here we use optical tweezers to track microtubule polymerization against microfabricated barriers, permitting unprecedented spatial resolution. RESULTS We find that microtubules exhibit extensive nanometer-scale variability in growth rate and often undergo shortening excursions, in some cases exceeding five tubulin layers, during periods of overall net growth. This result indicates that the guanosine triphosphate (GTP) cap does not exist as a single layer as previously proposed. We also find that length increments (over 100 ms time intervals, n = 16,762) are small, 0.81 +/- 6.60 nm (mean +/- standard deviation), and very rarely exceed 16 nm (about two dimer lengths), indicating that assembly occurs almost exclusively via single-subunit addition rather than via oligomers as was recently suggested. Finally, the assembly rate depends only weakly on load, with the average growth rate decreasing only 2-fold as the force increases 7-fold from 0.4 pN to 2.8 pN. CONCLUSIONS The data are consistent with a mechanochemical model in which a spatially extended GTP cap allows substantial shortening on the nanoscale, while still preventing complete catastrophe in most cases.

Rapid Microtubule Self-Assembly Kinetics

Melissa K. Gardner,1,2,3 Blake D. Charlebois,4 Imre M. Jánosi,5 Jonathon Howard,2 Alan J. Hunt,4,* and David J. Odde1,* 1Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA 2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 3Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA 5Department of Physics of Complex Systems, Loránd Eötvös University, Budapest, Hungary *Correspondence: ajhunt@umich.edu (A.J.H.), oddex002@umn.edu (D.J.O.) DOI 10.1016/j.cell.2011.06.053Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit dissociation rate from a microtubule tip is independent of free subunit concentration. Total-Internal-Reflection-Fluorescence (TIRF) microscopy experiments and data from a laser tweezers assay that measures in vitro microtubule assembly with nanometer resolution, provides evidence that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. We find that because both the association and the dissociation rates increase at higher free subunit concentrations, the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature.

Polarity in Stem Cell Division: Asymmetric Stem Cell Division in Tissue Homeostasis

Many adult stem cells divide asymmetrically to balance self-renewal and differentiation, thereby maintaining tissue homeostasis. Asymmetric stem cell divisions depend on asymmetric cell architecture (i.e., cell polarity) within the cell and/or the cellular environment. In particular, as residents of the tissues they sustain, stem cells are inevitably placed in the context of the tissue architecture. Indeed, many stem cells are polarized within their microenvironment, or the stem cell niche, and their asymmetric division relies on their relationship with the microenvironment. Here, we review asymmetric stem cell divisions in the context of the stem cell niche with a focus on Drosophila germ line stem cells, where the nature of niche-dependent asymmetric stem cell division is well characterized.

Advanced optical tweezers for the study of cellular and molecular biomechanics

Optical tweezers are an important tool for studying cellular and molecular biomechanics. We present a robust optical tweezers device with advanced features including: multiple optical traps, acousto-optic trap steering, and back focal plane interferometry position detection. We integrate these features into an upright microscope, with no compromise to its capabilities (differential interference contrast microscopy, fluorescence microscopy, etc.). Acousto-optic deflectors (AODs) steer each beam and can create multiple time-shared traps. Position detection, force calibrations and AOD performance are presented. The system can detect subnanometer displacements and forces below 0.1 pN.

Microtubule assembly dynamics: new insights at the nanoscale

Although the dynamic self-assembly behavior of microtubule ends has been well characterized at the spatial resolution of light microscopy (~200 nm), the single-molecule events that lead to these dynamics are less clear. Recently, a number of in vitro studies used novel approaches combining laser tweezers, microfabricated chambers, and high-resolution tracking of microtubule-bound beads to characterize mechanochemical aspects of MT dynamics at nanometer scale resolution. In addition, computational modeling is providing a framework for integrating these experimental results into physically plausible models of molecular scale microtubule dynamics. These nanoscale studies are providing new fundamental insights about microtubule assembly, and will be important for advancing our understanding of how microtubule dynamic instability is regulated in vivo via microtubule-associated proteins, therapeutic agents, and mechanical forces.

Self‐Assembled Magnetic Bead Biosensor for Measuring Bacterial Growth and Antimicrobial Susceptibility Testing

Bacterial antibiotic resistance is one of the major concerns of modern healthcare worldwide, and the development of rapid, growth-based, antimicrobial susceptibility tests is key for addressing it. The cover image shows a self-assembled asynchronous magnetic bead rotation (AMBR) biosensor developed for rapid detection of bacterial growth. Using the biosensors, the minimum inhibitory concentration of a clinical E. coli isolate can be measured within two hours, where currently tests take 6-24 hours. A 16-well prototype is also constructed for simple and robust observation of the self-assembled AMBR biosensors.

The Distribution of Polar Ejection Forces Determines the Amplitude of Chromosome Directional Instability

BACKGROUND Polar ejection forces have often been hypothesized to guide directional instability of mitotic chromosomes, but a direct link has never been established. This has led, in part, to the resurgence of alternative theories. By taking advantage of extremely precise femtosecond pulsed laser microsurgery, we abruptly alter the magnitude of polar ejection forces by severing vertebrate chromosome arms. RESULTS Reduction of polar ejection forces increases the amplitude of directional instability without altering other characteristics, thus establishing a direct link between polar ejection forces and the direction of chromosome movements. We find that polar ejection forces limit the range of chromosome oscillations by increasing the probability that motors at a leading kinetochore abruptly disengage or turn off, leading to a direction reversal. CONCLUSIONS From the relation between the change in oscillation amplitude and the amount a chromosome arm is shortened, we are able to map the distribution of polar ejection forces across the spindle, which is surprisingly different from previously assumed distributions. These results allow us to differentiate between the mechanisms proposed to underlie the directional instability of chromosomes.

Ultrafast laser fabrication of submicrometer pores in borosilicate glass

We demonstrate rapid fabrication of submicrometer-diameter pores in borosilicate glass using femtosecond laser machining and subsequent wet-etch techniques. This approach allows direct and repeatable fabrication of high-quality pores with diameters of 400-800 nm. Such small pores coupled with the desirable electrical and chemical properties of glass enable sensitive resistive-pulse analysis to determine the size and concentration of macromolecules and nanoparticles. Plasma-enhanced chemical vapor deposition allows further reduction of pore diameters to below 300 nm.

Kinetic properties of ASC protein aggregation in epithelial cells.

Apoptosis‐associated speck‐like protein with CARD domain (ASC), an adaptor protein composed of caspase recruitment and pyrin domains, can efficiently self‐associate to form a large spherical structure, called a speck. Although ASC aggregation is generally involved with both inflammatory processes and apoptosis, the detailed dynamics of speck formation have not been characterized. In this report, speck formation in HeLa cells transfected with ASC is examined by time‐lapse live‐imaging by confocal laser scanning microscopy. The results show that ASC aggregation is a very rapid and tightly regulated process. Prior to speck formation, soluble ASC aggregation is a low probability event, and the affinity of ASC subunits for one another is very low. Following a speck nucleation event, the affinity for further addition of ASC subunits increases dramatically, and aggregation is a highly energetically favorable reaction (Gibbs free energy ∼ −40 kJ/mol). This leads to a rapid depletion of soluble ASC, making it highly unlikely that a second speck will form inside the same cell and assuring that speck formation is “all or none,” with a well‐defined end point. Comparison with kinetic models of the aggregation process indicates diffusion, instead of active transport, is the dominant process for speck growth. Though speck formation and aggresome formation share some properties, we show that the two processes are distinct. J. Cell. Physiol. 222: 738–747, 2010.