Cytokine receptor-Eb1 interaction couples cell polarity and fate during asymmetric cell division.

Asymmetric stem cell division is a critical mechanism for balancing self-renewal and differentiation. Adult stem cells often orient their mitotic spindle to place one daughter inside the niche and the other outside of it to achieve asymmetric division. It remains unknown whether and how the niche may direct division orientation.

Dual-pore glass chips for cell-attached single-channel recordings.

While high-throughput planar patch-clamp instruments are now established to perform whole-cell recordings for drug screening, the conventional micropipette-based approach remains the gold standard for performing cell-attached single-channel recordings. Generally, planar platforms are not well-suited for such studies due to excess noise resulting from low seal resistances and the use of substrates with poor dielectric properties. Since these platforms tend to use the same pore to position a cell by suction and establish a seal, biological debris from the cell suspension can contaminate the pore surface prior to seal formation, reducing the seal resistance.

Rapid microtubule self-assembly kinetics.

Microtubule 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.

Spindle pole mechanics studied in mitotic asters: dynamic distribution of spindle forces through compliant linkages.

During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored.

Asymmetric division of cyst stem cells in Drosophila testis is ensured by anaphase spindle repositioning.

Many stem cells divide asymmetrically to balance self-renewal and differentiation. In Drosophila testes, two stem cell populations, germline stem cells (GSCs) and somatic cyst stem cells (CySCs), cohere and regulate one another. Here, we report that CySCs divide asymmetrically through repositioning the mitotic spindle around anaphase.

High-aspect ratio nanochannel formation by single femtosecond laser pulses.

Single femtosecond pulsed laser damage can be confined radially to regions smaller than the focus spot size due to the highly nonlinear mechanisms for energy absorption and ablation in transparent dielectrics. Along the propagation axis, however, we show that channels can be machined much deeper than the Rayleigh range of the laser focus. Using focused ion beam cross sections and acetate imprints, we analyze these channels and show that spherical aberration is not the primary source for this elongated damage, which is likely caused by microscale filamentation.

Liquid glass electrodes for nanofluidics.

Nanofluidic devices make use of molecular-level forces and phenomena to increase their density, speed and accuracy. However, fabrication is challenging, because dissimilar materials need to be integrated in three dimensions with nanoscale precision. Here, we report a three-dimensional nanoscale liquid glass electrode made from monolithic substrates without conductive materials by femtosecond-laser nanomachining.

Nanometer-resolution microtubule polymerization assays using optical tweezers and microfabricated barriers.

Microtubule (MT) polymerization dynamics, which are crucial to eukaryotic life and are the target of important anticancer agents, result from the addition and loss of 8-nm-long tubulin-dimer subunits. Addition and loss of one or a few subunits cannot be observed at the spatiotemporal resolution of conventional microscopy, and requires development of approaches with higher resolution. Here we describe an assay in which one end of an MT abuts a barrier, and MT length changes are coupled to the movement of an optically trapped bead, the motion of which is tracked with high resolution.

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.

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.

Time-lapse live imaging of stem cells in Drosophila testis.

This unit describes a protocol for time-lapse live-imaging of stem cells in Drosophila testis. Testis tips are dissected from Drosophila, sliced, and transferred to glass-bottom chambers where the stem cells residing in their native microenvironment can be monitored in real time. This protocol, facilitated with various fluorescence-labeled markers, allows dynamic cellular processes in stem cells to be characterized throughout the cell cycle.

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.

Novel morphologies and non-linear scaling of laser damage in glass by tightly-focused femtosecond pulses.

We examine the relationship between pulse energy and the morphology of damage in glass, produced by a tightly-focused femtosecond pulsed laser. For fluences up to three times that of threshold, an unexpected discontinuity in the scaling of damage size is caused by ejection of rings of material surrounding central damage that appear above a sharp threshold fluence. A mechanism for the production of these structures via thermal expansion and shockwave generation is proposed.

Minimal plus-end tracking unit of the cytoplasmic linker protein CLIP-170.

Cytoplasmic linker protein 170 (CLIP-170) is the prototype microtubule (MT) plus-end tracking protein (+TIP) and is involved in regulating MT dynamics. A comprehensive understanding of the process by which CLIP-170 tracks MT plus ends would provide insight into its function. However, the precise molecular mechanism of CLIP-170 +TIP behavior is unknown, and many potential models have been presented.

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.

Water-assisted femtosecond laser machining of electrospray nozzles on glass microfluidic devices.

Using water-assisted femtosecond laser machining, we fabricated electrospray nozzles on glass coverslips and on assembled microfluidic devices. Machining the nozzles after device assembly facilitated alignment of the nozzles over the microchannels. The basic nozzle design is a through-hole in the coverslip to pass liquids and a trough machined around the through-hole to confine the electrospray and prevent liquid from wicking across the glass surface.

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.

The motor activity of mammalian axonemal dynein studied in situ on doublet microtubules.

Flagellar dynein generates forces that produce relative shearing between doublet microtubules in the axoneme; this drives propagated bending of flagella and cilia. To better understand dynein's role in coordinated flagellar and ciliary motion, we have developed an in situ assay in which polymerized single microtubules glide along doublet microtubules extruded from disintegrated bovine sperm flagella at a pH of 7.8.

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.

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.

Microtubule movements on the arms of mitotic chromosomes: polar ejection forces quantified in vitro.

During mitosis, "polar ejection forces" (PEFs) are hypothesized to direct prometaphase chromosome movements by pushing chromosome arms toward the spindle equator. PEFs are postulated to be caused by (i) plus-end-directed microtubule (MT)-based motor proteins on the chromosome arms, namely chromokinesins, and (ii) the polymerization of spindle MTs into the chromosome. However, the exact role of PEFs is unclear, because little is known about their magnitude or their forms (e.