Cingulin and paracingulin tether myosins-2 to junctions to mechanoregulate the plasma membrane.

The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.

Collective dynein transport of the nucleus by pulling on astral microtubules during Saccharomyces cerevisiae mitosis.

Positioning the nucleus at the bud neck during Saccharomyces cerevisiae mitosis involves pulling forces of cytoplasmic dynein localized in the daughter cell. Although genetic analysis has revealed a complex network positioning the nucleus, quantification of the forces acting on the nucleus and the number of dyneins driving the process has remained difficult. To better understand the collective forces involved in nuclear positioning, we compare a model of dyneins-driven microtubule (MT) pulling, MT pushing, and cytoplasmic drag to experiments. During S. cerevisiae mitosis, MTs interacting with the cortex nucleated by the daughter spindle pole body (SPB) (SPB-D) are longer than the mother SPB (SPB-M), increasing further during spindle elongation in anaphase. Interphasic SPB mobility is effectively diffusive, while the mitotic mobility is directed. By optimizing a computational model of the mobility of the nucleus due to diffusion and MTs pushing at the cell membrane to experiment, we estimate the viscosity governing the drag force on nuclei during positioning. A force balance model of mitotic SPB mobility compared to experimental mobility suggests that even one or two dynein dimers are sufficient to move the nucleus in the bud neck. Using stochastic computer simulations of a budding cell, we find that punctate dynein localization can generate sufficient force to reel in the nucleus to the bud neck. Compared to uniform motor localization, puncta involve fewer motors suggesting a functional role for motor clustering. Stochastic simulations also suggest that a higher number of force generators than predicted by force balance may be required to ensure the robustness of spindle positioning.

Polymerization kinetics of tubulin from mung seedlings modeled as a competition between nucleation and GTP-hydrolysis rates.

Microtubules (MTs) form physiologically important cytoskeletal structures that are assembled by tubulin polymerization in nucleation- and guanosine triphosphate (GTP)-dependent manner. GTP hydrolysis competes with the addition of monomers, to determine the GTP-cap size, and the onset of shrinkage, which alternates with growth. Multiple theoretical models of MT polymerization dynamics have been reconciled to the kinetics of animal brain tubulins, but more recently, rapid kinetics seen in Arabidopsis tubulin polymerization suggest the need to sample a wider diversity in tubulin polymerization kinetics and reconcile it to theory. Here, we isolated tubulin from seedlings of Vigna sp. (mung bean), compared polymerization kinetics to animal brain tubulin, and used a computational model to understand the differences. We find that activity-isolated mung tubulin polymerizes in a nucleation-dependent manner, based on turbidimetry, qualitatively similar to brain tubulin, but with a 10-fold smaller critical concentration. GTP-dependent polymerization kinetics also appear to be transient, indicative of high rates of GTP hydrolysis. Computational modeling of tubulin nucleation and vectorial GTP hydrolysis to examine the effects of high nucleation and GTP-hydrolysis rates predicts a dominance of the latter in determining MT lengths and numbers. Microscopy of mung tubulin filaments stabilized by GMPCPP or taxol results in few and short MTs, compared to the many long MTs arising from goat tubulin, qualitatively matching the model predictions. We find GTP-hydrolysis outcompetes nucleation rates in determining MT lengths and numbers.

Collective effects of yeast cytoplasmic dynein based microtubule transport.

Teams of cortically anchored dyneins pulling at microtubules (MTs) are known to be essential for aster, spindle and nuclear positioning during cell division and fertilization. While the single-molecule basis of dynein processivity is now better understood, the effect of increasing numbers of motors on transport is not clear. Here, we examine the collective transport properties of a Saccharomyces cerevisiae cytoplasmic dynein fragment, widely used as a minimal model, by a combination of quantitative MT gliding assays and stochastic simulations. We find both MT lengths and motor densities qualitatively affect the degree of randomness of MT transport. However, the directionality and velocity of MTs increase above a threshold number of motors (N) interacting with a filament. To better understand this behavior, we simulate a gliding assay based on a model of uniformly distributed immobilized motors transporting semi-flexible MTs. Each dynein dimer is modeled as an effective stochastic stepper with asymmetric force dependent detachment dynamics, based on single-molecule experiments. Simulations predict increasing numbers of motors (N) result in a threshold dependent transition in directionality and transport velocity and a monotonic decrease in effective diffusivity. Thus both experiment and theory show qualitative agreement in the emergence of coordination in transport above a threshold number of motor heads. We hypothesize that the phase-transition like property of this dynein could play a role in vivo during yeast mitosis, when this dynein localizes to the cortex and pulls astral MTs of increasing length, resulting in correct positioning and orientation of the nucleus at the bud-neck.

Automated Multi-Peak Tracking Kymography (AMTraK): A Tool to Quantify Sub-Cellular Dynamics with Sub-Pixel Accuracy.

Kymographs or space-time plots are widely used in cell biology to reduce the dimensions of a time-series in microscopy for both qualitative and quantitative insight into spatio-temporal dynamics. While multiple tools for image kymography have been described before, quantification remains largely manual. Here, we describe a novel software tool for automated multi-peak tracking kymography (AMTraK), which uses peak information and distance minimization to track and automatically quantify kymographs, integrated in a GUI. The program takes fluorescence time-series data as an input and tracks contours in the kymographs based on intensity and gradient peaks. By integrating a branch-point detection method, it can be used to identify merging and splitting events of tracks, important in separation and coalescence events. In tests with synthetic images, we demonstrate sub-pixel positional accuracy of the program. We test the program by quantifying sub-cellular dynamics in rod-shaped bacteria, microtubule (MT) transport and vesicle dynamics. A time-series of E. coli cell division with labeled nucleoid DNA is used to identify the time-point and rate at which the nucleoid segregates. The mean velocity of microtubule (MT) gliding motility due to a recombinant kinesin motor is estimated as 0.5 µm/s, in agreement with published values, and comparable to estimates using software for nanometer precision filament-tracking. We proceed to employ AMTraK to analyze previously published time-series microscopy data where kymographs had been manually quantified: clathrin polymerization kinetics during vesicle formation and anterograde and retrograde transport in axons. AMTraK analysis not only reproduces the reported parameters, it also provides an objective and automated method for reproducible analysis of kymographs from in vitro and in vivo fluorescence microscopy time-series of sub-cellular dynamics.