Erkan Tüzel

Myosin XI Is Essential for Tip Growth in Physcomitrella patens

Class XI myosins are unique to plants and known to function in cytoplasmic streaming. This study demonstrates that the two myosin XI genes present in the moss Physcomitrella patens are functionally redundant, localize to the apical region of actively growing protonemal cells, and play a key role in tip growth. Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5′ untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells.

Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Microtubules (MTs) have been proposed to act mechanically as compressive struts that resist both actomyosin contractile forces and their own polymerization forces to mechanically stabilize cell shape. To identify the origin of MT bending, we directly observed MT bending and F-actin transport dynamics in the periphery of LLC-PK1 epithelial cells. We found that F-actin is nearly stationary in these cells even as MTs are deformed, demonstrating that MT bending is not driven by actomyosin contractility. Furthermore, the inhibition of myosin II activity through the use of blebbistatin results in microtubules that are still dynamically bending. In addition, as determined by fluorescent speckle microscopy, MT polymerization rarely results, if ever, in bending. We suppressed dynamic instability using nocodazole, and we observed no qualitative change in the MT bending dynamics. Bending most often results from anterograde transport of proximal portions of the MT toward a nearly stationary distal tip. Interestingly, we found that in an in vitro kinesin-MT gliding assay, MTs buckle in a similar manner. To make quantitative comparisons, we measured curvature distributions of observed MTs and found that the in vivo and in vitro curvature distributions agree quantitatively. In addition, the measured MT curvature distribution is not Gaussian, as expected for a thermally driven semiflexible polymer, indicating that thermal forces play a minor role in MT bending. We conclude that many of the known mechanisms of MT deformation, such as polymerization and acto-myosin contractility, play an inconsequential role in mediating MT bending in LLC-PK1 cells and that MT-based molecular motors likely generate most of the strain energy stored in the MT lattice. The results argue against models in which MTs play a major mechanical role in LLC-PK1 cells and instead favor a model in which mechanical forces control the spatial distribution of the MT array.

Transport coefficients for stochastic rotation dynamics in three dimensions.

Explicit expressions for the transport coefficients of a recently introduced stochastic model for simulating fluctuating fluid dynamics are derived in three dimensions by means of Green-Kubo relations and simple kinetic arguments. The results are shown to be in excellent agreement with simulation data. Two collision rules are considered and their computational efficiency is compared.

Actin Interacting Protein1 and Actin Depolymerizing Factor Drive Rapid Actin Dynamics in Physcomitrella patens

This study evaluates the effect of two actin binding proteins (AIP1 and ADF) on F-actin dynamics in moss. Whereas AIP1 enhances F-actin turnover, ADF is required for it. The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics.

Motor transport of self-assembled cargos in crowded environments

Intracellular transport of cargo particles is performed by multiple motors working in concert. However, the mechanism of motor association to cargos is unknown. It is also unknown how long individual motors stay attached, how many are active, and how multimotor cargos would navigate a densely crowded filament with many other motors. Prior theoretical and experimental biophysical model systems of intracellular cargo have assumed fixed teams of motors transporting along bare microtubules or microtubules with fixed obstacles. Here, we investigate a regime of cargos transporting along microtubules crowded with free motors. Furthermore, we use cargos that are able to associate or dissociate motors as it translocates. We perform in vitro motility reconstitution experiments with high-resolution particle tracking. Our model system consists of a quantum dot cargo attached to kinesin motors, and additional free kinesin motors that act as traffic along the microtubule. Although high densities of kinesin motors hinder forward motion, resulting in a lower velocity, the ability to associate motors appears to enhance the run length and attachment time of the quantum dot, improving overall cargo transport. These results suggest that cargos that can associate new motors as they transport could overcome traffic jams.

Analysis of Microtubule Curvature

The microtubule cytoskeleton in living cells generate and resist mechanical forces to mediate fundamental cell processes, including cell division and migration. Recent advances in digital fluorescence microscopy have enabled the direct observation of bending of individual microtubules in living cells, which has enabled quantitative estimation of the mechanical state of the microtubule array. Although a variety of mechanisms have been proposed, the precise origins of microtubule deformation in living cells remain largely obscure. To investigate these mechanisms and their relative importance in cellular processes, a method is needed to accurately quantify microtubule bending within living cells. Here we describe a method for quantification of bending, using digital fluorescence microscope images to estimate the distribution of curvature in the microtubule. Digital images of individual microtubules can be used to obtain a set of discrete x-y coordinates along the microtubule contour, which is then used to estimate the curvature distribution. Due to system noise and digitization error, the estimate will be inaccurate to some degree. To quantify the inaccuracy, a computational model is used to simulate both the bending of thermally driven microtubules and their observation by digital fluorescence microscopy. This allows for direct comparison between experimental and simulated images, a method which we call model convolution microscopy. We assess the accuracy of various methods and present a suitable method for estimating the curvature distribution for thermally driven semiflexible polymers. Finally, we discuss extensions of the method to quantify microtubule curvature in living cells.

Equilibrium calculation of transport coefficients for a fluid-particle model

A recently introduced particle-based model for fluid flow, called stochastic rotation dynamics, can be made Galilean invariant by introducing a random shift of the computational grid before collisions. In this paper, it is shown how the Green-Kubo relations derived previously can be resummed to obtain exact expressions for the collisional contributions to the transport coefficients. It is also shown that the collisional contribution to the microscopic stress tensor is not symmetric, and that this leads to an additional viscosity. The resulting identification of the transport coefficients for the hydrodynamic modes is discussed in detail, and it is shown that this does not impose restrictions on the applicability of the model. The collisional contribution to the thermal conductivity, which becomes important for small mean free path and small average particle number per cell, is also derived.

Exhaustion of racing sperm in nature-mimicking microfluidic channels during sorting.

Fertilization is central to the survival and propagation of a species, however, the precise mechanisms that regulate the sperms journey to the egg are not well understood. In nature, the sperm has to swim through the cervical mucus, akin to a microfluidic channel. Inspired by this, a simple, cost-effective microfluidic channel is designed on the same scale. The experimental results are supported by a computational model incorporating the exhaustion time of sperm.

Dynamic correlations in stochastic rotation dynamics

The dynamic structure factor, vorticity and entropy density dynamic correlation functions are measured for stochastic rotation dynamics (SRD), a particle based algorithm for fluctuating fluids. This allows us to obtain unbiased values for the longitudinal transport coefficients such as thermal diffusivity and bulk viscosity. The results are in good agreement with earlier numerical and theoretical results, and it is shown for the first time that the bulk viscosity is indeed zero for this algorithm. In addition, corrections to the self-diffusion coefficient and shear viscosity arising from the breakdown of the molecular chaos approximation at small mean free paths are analyzed. In addition to deriving the form of the leading correlation corrections to these transport coefficients, the probabilities that two and three particles remain collision partners for consecutive time steps are derived analytically in the limit of small mean free path. The results of this paper verify that we have an excellent understanding of the SRD algorithm at the kinetic level and that analytic expressions for the transport coefficients derived elsewhere do indeed provide a very accurate description of the SRD fluid.

Transport by Populations of Fast and Slow Kinesins Uncovers Novel Family-Dependent Motor Characteristics Important for In Vivo Function

Intracellular cargo transport frequently involves multiple motor types, either having opposite directionality or having the same directionality but different speeds. Although significant progress has been made in characterizing kinesin motors at the single-molecule level, predicting their ensemble behavior is challenging and requires tight coupling between experiments and modeling to uncover the underlying motor behavior. To understand how diverse kinesins attached to the same cargo coordinate their movement, we carried out microtubule gliding assays using pairwise mixtures of motors from the kinesin-1, -2, -3, -5, and -7 families engineered to have identical run lengths and surface attachments. Uniform motor densities were used and microtubule gliding speeds were measured for varying proportions of fast and slow motors. A coarse-grained computational model of gliding assays was developed and found to recapitulate the experiments. Simulations incorporated published force-dependent velocities and run lengths, along with mechanical interactions between motors bound to the same microtubule. The simulations show that the force-dependence of detachment is the key parameter that determines gliding speed in multimotor assays, while motor compliance, surface density, and stall force all play minimal roles. Simulations also provide estimates for force-dependent dissociation rates, suggesting that kinesin-1 and the mitotic motors kinesin-5 and -7 maintain microtubule association against loads, whereas kinesin-2 and -3 readily detach. This work uncovers unexpected motor behavior in multimotor ensembles and clarifies functional differences between kinesins that carry out distinct mechanical tasks in cells.