Microtubule Length Distribution Research Articles

Minimal Mechanisms of Microtubule Length Regulation in Living Cells.

The microtubule cytoskeleton is responsible for sustained, long-range intracellular transport of mRNAs, proteins, and organelles in neurons. Neuronal microtubules must be stable enough to ensure reliable transport, but they also undergo dynamic instability, as their plus and minus ends continuously switch between growth and shrinking. This process allows for continuous rebuilding of the cytoskeleton and for flexibility in injury settings. Motivated by in vivo experimental data on microtubule behavior in Drosophila neurons, we propose a mathematical model of dendritic microtubule dynamics, with a focus on understanding microtubule length, velocity, and state-duration distributions. We find that limitations on microtubule growth phases are needed for realistic dynamics, but the type of limiting mechanism leads to qualitatively different responses to plausible experimental perturbations. We therefore propose and investigate two minimally-complex length-limiting factors: limitation due to resource (tubulin) constraints and limitation due to catastrophe of large-length microtubules. We combine simulations of a detailed stochastic model with steady-state analysis of a mean-field ordinary differential equations model to map out qualitatively distinct parameter regimes. This provides a basis for predicting changes in microtubule dynamics, tubulin allocation, and the turnover rate of tubulin within microtubules in different experimental environments.

Dynamics and length distributions of microtubules with a multistep catastrophe mechanism

Regarding the experimental observation that microtubule (MT) catastrophe can be described as a multistep process, we extend the Dogterom–Leibler model for dynamic instability in order to discuss the effect that such a multistep catastrophe mechanism has on the distribution of MT lengths in the two regimes of bounded and unbounded growth. We show that in the former case, the steady state length distribution is non-exponential and has a lighter tail if multiple steps are required to undergo a catastrophe. If rescue events are possible, we detect a maximum in the distribution, i.e. the MT has a most probable length greater than zero. In the regime of unbounded growth, the length distribution converges to a Gaussian distribution whose variance decreases with the number of catastrophe steps. We extend our work by applying the multistep catastrophe model to MTs that grow against an opposing force and to MTs that are confined between two rigid walls. We determine critical forces below which the MT is in the bounded regime, and show that the multistep characteristics of the length distribution are largely lost if the growth of an MT in the unbounded regime is restricted by a rigid wall. All results are verified by stochastic simulations.

Microtubule organization and cell geometry.

A characteristic feature of nondividing animal cells is the radial organization of microtubules (MTs), emanating from a single microtubule organizing center (MTOC). As generically these cells are not spherically symmetric, this raises the question of the influence of cell geometry on the orientational distribution of microtubules. We present a systematic study of this question in a simplified setting where MTs are nucleated from a single fixed MTOC in the center of an elliptical cell geometry. Within this context we introduce four models of increasing complexity, each one introducing additional mechanisms that govern the interaction of the MTs with the cell boundary. In order, we consider the cases: MTs that can bind to the boundary with a fixed mean residence time (M0), force-producing MTs that can slide on the boundary towards the cell poles (MS), MTs that interact with a generic polarity factor that is transported and deposited at the boundary, and which in turn stabilizes the MTs at the boundary (MP), and a final model in which both sliding and stabilization by polarity factors is taken into account (MSP). In the baseline model (M0), the exponential length distribution of MTs causes most of the interactions at the cell boundary to occur along the shorter transverse direction in the cell, leading to transverse biaxial order. MT sliding (MS) is able to reorient the main axis of this biaxial order along the longitudinal axis. The polarization mechanism introduced in MP and MSP overrules the geometric bias towards bipolar order observed in M0 and MS, and allows the establishment of unipolar order either along the short (MP) or the long cell axis (MSP). The behavior of the latter two models can be qualitatively reproduced by a very simple toy model with discrete MT orientations.

Super-resolution imaging and quantitative analysis of microtubule arrays in model neurons show that epothilone D increases the density but decreases the length and straightness of microtubules in axon-like processes

Microtubules are essential for the development of neurons and the regulation of their structural plasticity. Microtubules also provide the structural basis for the long-distance transport of cargo. Various factors influence the organization and dynamics of neuronal microtubules, and disturbance of microtubule regulation is thought to play a central role in neurodegenerative diseases. However, imaging and quantitative assessment of the microtubule organization in the densely packed neuronal processes is challenging. The development of super-resolution techniques combined with the use of nanobodies offers new possibilities to visualize microtubules in neurites in high resolution. In combination with recently developed computational analysis tools, this allows automated quantification of neuronal microtubule organization with high precision. Here we have implemented three-dimensional DNA-PAINT (Point Accumulation in Nanoscale Topography), a single-molecule localization microscopy (SMLM) technique, which allows us to acquire 3D arrays of the microtubule lattice in axons of model neurons (neuronally differentiated PC12 cells) and dendrites of primary neurons. For the quantitative analysis of the microtubule organization, we used the open-source software package SMLM image filament extractor (SIFNE). We found that treatment with nanomolar concentrations of the microtubule-targeting drug epothilone D (EpoD) increased microtubule density in axon-like processes of model neurons and shifted the microtubule length distribution to shorter ones, with a mean microtubule length of 2.39 µm (without EpoD) and 1.98 µm (with EpoD). We also observed a significant decrease in microtubule straightness after EpoD treatment. The changes in microtubule density were consistent with live-cell imaging measurements of ensemble microtubule dynamics using a previously established Fluorescence Decay After Photoactivation (FDAP) assay. For comparison, we determined the organization of the microtubule array in dendrites of primary hippocampal neurons. We observed that dendritic microtubules have a very similar length distribution and straightness compared to microtubules in axon-like processes of a neuronal cell line. Our data show that super-resolution imaging of microtubules followed by algorithm-based image analysis represents a powerful tool to quantitatively assess changes in microtubule organization in neuronal processes, useful to determine the effect of microtubule-modulating conditions. We also provide evidence that the approach is robust and can be applied to neuronal cell lines or primary neurons, both after incorporation of labeled tubulin and by anti-tubulin antibody staining.

Microtubule reorganization during female meiosis in C. elegans.

Most female meiotic spindles undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of meiotic spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown. To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic Caenorhabditis elegans spindles. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over within seconds. The results show that the metaphase to anaphase transition correlates with an increase in microtubule numbers and a decrease in their average length. Detailed analysis of the tomographic data revealed that the microtubule length changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the spindle reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe. This suggests that the most prominent drivers of spindle rearrangements are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on katanin.

Effects of Severing Enzymes on the Length Distribution and Total Mass of Microtubules

Microtubules are cytoskeletal polymers of αβ-tubulin whose dynamics is highly regulated as eukaryotic cells undergo cell division, migration and tissue development. Microtubule severing enzymes, spastin, katanin and fidgetin use ATP-hydrolysis to fragment microtubules into shorter filaments. Paradoxically, in vivo experiments have shown that severases can increase total microtubule number and mass inside the cells, suggesting that severases possess a nucleation-like activity. We have resolved this paradox by reconstituting a dynamic microtubule severing system in vitro and discovered that Drosophila spastin has two additional properties in addition to severing: it increases microtubule rescue and slows down shrinkage. Under the right circumstances these effects lead to an exponential increase in the number and mass of microtubules. In this abstract, we address the question of how the exponential rate constant and the length distribution depend on severing and microtubule dynamics. A mathematical model reveals that the amplification rate depends on the balance of creating new microtubules through severing and the loss of existing microtubule through shrinkage. The mean length depends on the ratio of the growth rate to the severing rate and can be well-approximated by a simplified no-catastrophe model, which we have solved analytically. Unexpectedly, dynamic instability has only a small impact on microtubule length. On the other hand, dynamic instability can influence the amplification rate. Thus differential regulation of the dynamic parameters can separately modulate different aspects of the microtubule network. Our results establish the quantitative basis for how severing and dynamics conjunctly constitute a versatile mechanism to regulate microtubule length and mass in the complex cellular environment.

Predicted Effects of Severing Enzymes on the Length Distribution and Total Mass of Microtubules

Microtubules are dynamic cytoskeletal polymers whose growth and shrinkage are highly regulated as eukaryotic cells change shape, move, and divide. One family of microtubule regulators includes the ATP-hydrolyzing enzymes spastin, katanin, and fidgetin, which sever microtubule polymers into shorter fragments. Paradoxically, severases can increase microtubule number and mass in cells. Recent work with purified spastin and katanin accounts for this phenotype by showing that, in addition to severing, these enzymes modulate microtubule dynamics by accelerating the conversion of microtubules from their shrinking to their growing states and thereby promoting their regrowth. This leads to the observed exponential increase in microtubule mass. Spastin also influences the steady-state distribution of microtubule lengths, changing it from an exponential, as predicted by models of microtubule dynamic instability, to a peaked distribution. This effect of severing and regrowth by spastin on the microtubule length distribution has not been explained theoretically. To solve this problem, we formulated and solved a master equation for the time evolution of microtubule lengths in the presence of severing and microtubule dynamic instability. We then obtained numerical solutions to the steady-state length distribution and showed that the rate of severing and the speed of microtubule growth are the dominant parameters determining the steady-state length distribution. Furthermore, we found that the amplification rate is predicted to increase with severing, which is, to our knowledge, a new result. Our results establish a theoretical basis for how severing and dynamics together can serve to nucleate new microtubules, constituting a versatile mechanism to regulate microtubule length and mass.

A Growth-Fragmentation Approach for Modeling Microtubule Dynamic Instability.

Microtubules (MTs) are protein filaments found in all eukaryotic cells which are crucial for many cellular processes including cell movement, cell differentiation, and cell division. Due to their role in cell division, they are often used as targets for chemotherapy drugs used in cancer treatment. Experimental studies of MT dynamics have played an important role in the development and administration of many novel cancer drugs; however, a complete description of MT dynamics is lacking. Here, we propose a new mathematical model for MT dynamics, that can be used to study the effects of chemotherapy drugs on MT dynamics. Our model consists of a growth-fragmentation equation describing the dynamics of a length distribution of MTs, coupled with two ODEs that describe the dynamics of free GTP- and GDP-tubulin concentrations (the individual dimers that comprise of MTs). Here, we prove the well-posedness of our system and perform a numerical exploration of the influence of certain model parameters on the systems dynamics. In particular, we focus on a qualitative description for how a certain class of destabilizing drugs, the vinca alkaloids, alter MT dynamics. Through variation of certain model parameters which we know are altered by these drugs, we make comparisons between simulation results and what is observed in in vitro studies.

Monte Carlo simulations of microtubule arrays: The critical roles of rescue transitions, the cell boundary, and tubulin concentration in shaping microtubule distributions.

Microtubules are dynamic polymers required for a number of processes, including chromosome movement in mitosis. While regulators of microtubule dynamics have been well characterized, we lack a convenient way to predict how the measured dynamic parameters shape the entire microtubule system within a cell, or how the system responds when specific parameters change in response to internal or external signals. Here we describe a Monte Carlo model to simulate an array of dynamic microtubules from parameters including the cell radius, total tubulin concentration, microtubule nucleation rate from the centrosome, and plus end dynamic instability. The algorithm also allows dynamic instability or position of the cell edge to vary during the simulation. Outputs from simulations include free tubulin concentration, average microtubule lengths, length distributions, and individual length changes over time. Using this platform and reported parameters measured in interphase LLCPK1 epithelial cells, we predict that sequestering ~ 15–20% of total tubulin results in fewer microtubules, but promotes dynamic instability of those remaining. Simulations also predict that lowering nucleation rate will increase the stability and average length of the remaining microtubules. Allowing the position of the cell’s edge to vary over time changed the average length but not the number of microtubules and generated length distributions consistent with experimental measurements. Simulating the switch from interphase to prophase demonstrated that decreased rescue frequency at prophase is the critical factor needed to rapidly clear the cell of interphase microtubules prior to mitotic spindle assembly. Finally, consistent with several previous simulations, our results demonstrate that microtubule nucleation and dynamic instability in a confined space determines the partitioning of tubulin between monomer and polymer pools. The model and simulations will be useful for predicting changes to the entire microtubule array after modification to one or more parameters, including predicting the effects of tubulin-targeted chemotherapies.

Limited Resources Induce Bistability in Microtubule Length Regulation

The availability of protein is an important factor for the determination of the size of the mitotic spindle. Involved in spindle-size regulation is kinesin-8, a molecular motor and microtubule (MT) depolymerase, which is known to tightly control MT length. Here, we propose and analyze a theoretical model in which kinesin-induced MT depolymerization competes with spontaneous polymerization while supplies of both tubulin and kinesin are limited. In contrast to previous studies where resources were unconstrained, we find that, for a wide range of concentrations, MT length regulation is bistable. We test our predictions by conducting invitro experiments and find that the bistable behavior manifests in a bimodal MT length distribution.

Connecting macroscopic dynamics with microscopic properties in active microtubule network contraction

The cellular cytoskeleton is an active material, driven out of equilibrium by molecular motor proteins. It is not understood how the collective behaviors of cytoskeletal networks emerge from the properties of the network’s constituent motor proteins and filaments. Here we present experimental results on networks of stabilized microtubules in Xenopus oocyte extracts, which undergo spontaneous bulk contraction driven by the motor protein dynein, and investigate the effects of varying the initial microtubule density and length distribution. We find that networks contract to a similar final density, irrespective of the length of microtubules or their initial density, but that the contraction timescale varies with the average microtubule length. To gain insight into why this microscopic property influences the macroscopic network contraction time, we developed simulations where microtubules and motors are explicitly represented. The simulations qualitatively recapitulate the variation of contraction timescale with microtubule length, and allowed stress contributions from different sources to be estimated and decoupled.

Nonrandom γ-TuNA-dependent spatial pattern of microtubule nucleation at the Golgi.

Noncentrosomal microtubule (MT) nucleation at the Golgi generates MT network asymmetry in motile vertebrate cells. Investigating the Golgi-derived MT (GDMT) distribution, we find that MT asymmetry arises from nonrandom nucleation sites at the Golgi (hotspots). Using computational simulations, we propose two plausible mechanistic models of GDMT nucleation leading to this phenotype. In the "cooperativity" model, formation of a single GDMT promotes further nucleation at the same site. In the "heterogeneous Golgi" model, MT nucleation is dramatically up-regulated at discrete and sparse locations within the Golgi. While MT clustering in hotspots is equally well described by both models, simulating MT length distributions within the cooperativity model fits the data better. Investigating the molecular mechanism underlying hotspot formation, we have found that hotspots are significantly smaller than a Golgi subdomain positive for scaffolding protein AKAP450, which is thought to recruit GDMT nucleation factors. We have further probed potential roles of known GDMT-promoting molecules, including γ-TuRC-mediated nucleation activator (γ-TuNA) domain-containing proteins and MT stabilizer CLASPs. While both γ-TuNA inhibition and lack of CLASPs resulted in drastically decreased GDMT nucleation, computational modeling revealed that only γ-TuNA inhibition suppressed hotspot formation. We conclude that hotspots require γ-TuNA activity, which facilitates clustered GDMT nucleation at distinct Golgi sites.

Model of Growth Cone Membrane Polarization via Microtubule Length Regulation

We present a mathematical model of membrane polarization in growth cones. We proceed by coupling an active transport model of cytosolic proteins along a two-dimensional microtubule (MT) network with a modified Dogterom-Leibler model of MT growth. In particular, we consider a Rac1-stathmin-MT pathway in which the growth and catastrophe rates of MTs are regulated by cytosolic stathmin, while the stathmin is regulated by Rac1 at the membrane. We use regular perturbation theory and numerical simulations to determine the steady-state stathmin concentration, the mean MT length distribution, and the resulting distribution of membrane-bound proteins. We thus show how a nonuniform Rac1 distribution on the membrane generates a polarized distribution of membrane proteins. The mean MT length distribution and hence the degree of membrane polarization are sensitive to the precise form of the Rac1 distribution and parameters such as the catastrophe-promoting constant and tubulin association rate. This is a consequence of the fact that the lateral diffusion of stathmin tends to weaken the effects of Rac1 on the distribution of mean MT lengths.

Polymerization of microtubules in the presence of diffusing tubulins: a boundary layer analysis

Microtubules are biopolymers with an interesting growth dynamics that alternates between persistent phases of polymerization and depolymerization. The growth dynamics is expected to be influenced by the dynamics of the tubulin molecules around the microtubule. In this paper, we present a boundary layer based method that allows us to study systematically a nonlinear growth model coupling the growth dynamics of microtubule and the diffusive dynamics of free tubulins in the neighboring environment. Following previous experimental studies, we consider the growth and the rescue rates to be dependent on the tubulin density in the neighborhood of the microtubule tip. Considering the effect of various regulatory proteins that facilitate the motion of tubulin molecules along the direction of growth, we incorporate an effective biased diffusion of tubulin molecules. Based on the values of the bias velocity and other parameters, the boundary layer analysis shows two regions with distinct nature of microtubule length distributions. In order to show the wider applicability of the method, we briefly discuss the case of tubulin density dependent catastrophe rate. Some of our results are supported by numerical simulations.

Dissecting microtubule structures by laser ablation.

Here, we describe a detailed protocol, based on laser ablation and fluorescence optical microscopy, to measure the microtubule organization in spindles, including microtubule length distribution, polarity, and plus and minus end densities. The method uses the asymmetry in microtubule depolymerization after a cut, where the newly created microtubule plus ends depolymerize all the way to the minus ends, whereas the newly created minus ends remain stable. The protocol described in this chapter is optimized for spindles, but can be easily applied to any microtubule-based structure. The chapter is divided into two parts. First, we provide the theoretical basis for the method. Second, we describe in detail all steps necessary to reconstruct the microtubule organization of a spindle assembled in Xenopus laevis egg extract. Compared to electron microscopy, which in theory can resolve individual microtubules in spindles and provide similar structural information, our method is fast and simple enough to allow for a full quantitative reconstruction of the microtubule organization of several X. laevis spindles—which have volumes tens of thousands of times larger than spindles whose structures have been previously solved by electron microscopy—in a single experimental session, as well as to explore how the architecture of these structures changes in response to biochemical perturbations.

Feedback Mechanism for Microtubule Length Regulation by Stathmin Gradients

We formulate and analyze a theoretical model for the regulation of microtubule (MT) polymerization dynamics by the signaling proteins Rac1 and stathmin. In cells, the MT growth rate is inhibited by cytosolic stathmin, which, in turn, is inactivated by Rac1. Growing MTs activate Rac1 at the cell edge, which closes a positive feedback loop. We investigate both tubulin sequestering and catastrophe promotion as mechanisms for MT growth inhibition by stathmin. For a homogeneous stathmin concentration in the absence of Rac1, we find a switchlike regulation of the MT mean length by stathmin. For constitutively active Rac1 at the cell edge, stathmin is deactivated locally, which establishes a spatial gradient of active stathmin. In this gradient, we find a stationary bimodal MT-length distribution for both mechanisms of MT growth inhibition by stathmin. One subpopulation of the bimodal length distribution can be identified with fast-growing and long pioneering MTs in the region near the cell edge, which have been observed experimentally. The feedback loop is closed through Rac1 activation by MTs. For tubulin sequestering by stathmin, this establishes a bistable switch with two stable states: one stable state corresponds to upregulated MT mean length and bimodal MT length distributions, i.e., pioneering MTs; the other stable state corresponds to an interrupted feedback with short MTs. Stochastic effects as well as external perturbations can trigger switching events. For catastrophe-promoting stathmin, we do not find bistability.

Measurements of Single Fluorescent Motor Proteins: The Right Way

Cytoskeletal motor proteins are required in many cellular processes, such as intracellular transport and mitosis. Therefore, the biophysical characterization of motor protein movement along their filamentous tracks is essential. Commonly, stepping motility assays are used to determine the stepping and detachment rates of various molecular motor proteins by measuring their speed, run length and interaction time. However, comparison of these results proved to be difficult because the experimental setup (e.g. bead assay vs. single-molecule fluorescence assay), the experimental conditions (e.g. temperature, buffer or filament preparation) and data analysis (e.g. normal vs. exponential distribution) can influence the results. Here, we describe a method to evaluate traces of fluorescent motor proteins and propose an algorithm to correct the measurements for photobleaching and the limited length of the filaments. Additionally, bootstrapping is used to estimate statistical errors of the evaluation method. The method was tested with numerical simulations as well as with experimental data from kinesin-1 stepping experiments to show that the run length of kinesin-1 is independent of the microtubule length distribution. Our work will not only improve the evaluation of experimental data, but will also allow for better statistical comparison of two or more populations of motor proteins (e.g. motors with distinct mutations or motors linked to different cargos).

Single-Molecule Motility: Statistical Analysis and the Effects of Track Length on Quantification of Processive Motion

In vitro, single-molecule motility assays allow for the direct characterization of molecular motor properties including stepping velocity and characteristic run length. Although application of these techniques in vivo is feasible, the challenges involved in sample preparation, as well as the added complexity of the cell and its systems, result in a reduced ability to collect large datasets, as well as difficulty in simultaneous observation of the components of the motility system, namely motor and track. To address these challenges, we have developed simulations to characterize motility datasets as a function of sample size, processive run length of the motor, and distribution of track lengths. We introduce the use of a simple bootstrapping technique that allows for the quantification of measurement uncertainty and a Monte Carlo permutation resampling scheme for the measurement of statistical significance and the estimation of required sample size. In addition, we have found that, despite conventional wisdom, the measured characteristic run length is directly coupled to the characteristic track length that describes the microtubule length distribution. To be able to make comparisons between motility experiments performed on different track populations as well as make measurements of motility when motors and tracks cannot be simultaneously resolved, we have developed a theoretical framework for the determination of the effect that track length has on observed characteristic run lengths. This shows good agreement with in vitro motility experiments on two kinesin constructs walking on microtubule populations of different characteristic track lengths.

Dynamics and length distribution of microtubules under force and confinement

We investigate the microtubule polymerization dynamics with catastrophe and rescue events for three different confinement scenarios, which mimic typical cellular environments: (i) The microtubule is confined by rigid and fixed walls, (ii) it grows under constant force, and (iii) it grows against an elastic obstacle with a linearly increasing force. We use realistic catastrophe models and analyze the microtubule dynamics, the resulting microtubule length distributions, and force generation by stochastic and mean field calculations; in addition, we perform stochastic simulations. Freely growing microtubules exhibit a phase of bounded growth with finite microtubule length and a phase of unbounded growth. The main results for the three confinement scenarios are as follows: (i) In confinement by fixed rigid walls, we find exponentially decreasing or increasing stationary microtubule length distributions instead of bounded or unbounded phases, respectively. We introduce a realistic model for wall-induced catastrophes and investigate the behavior of the average length as a function of microtubule growth parameters. (ii) Under a constant force, the boundary between bounded and unbounded growth is shifted to higher tubulin concentrations and rescue rates. The critical force f(c) for the transition from unbounded to bounded growth increases logarithmically with tubulin concentration and the rescue rate, and it is smaller than the stall force. (iii) For microtubule growth against an elastic obstacle, the microtubule length and polymerization force can be regulated by microtubule growth parameters. For zero rescue rate, we find that the average polymerization force depends logarithmically on the tubulin concentration and is always smaller than the stall force in the absence of catastrophes and rescues. For a nonzero rescue rate, we find a sharply peaked steady-state length distribution, which is tightly controlled by microtubule growth parameters. The corresponding average microtubule length self-organizes such that the average polymerization force equals the critical force f(c) for the transition from unbounded to bounded growth. We also investigate the force dynamics if growth parameters are perturbed in dilution experiments. Finally, we show the robustness of our results against changes of catastrophe models and load distribution factors.

Electron tomography of the microtubule cytoskeleton in multinucleated hyphae of Ashbya gossypii

We report the mechanistic basis guiding the migration pattern of multiple nuclei in hyphae of Ashbya gossypii. Using electron tomography, we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the spindle microtubules of four mitotic nuclei. Each spindle pole body (SPB) nucleates three cMTs and most cMTs above a certain length grow according to their plus-end structure. Long cMTs closely align for several microns along the cortex, presumably marking regions where dynein generates pulling forces on nuclei. Close proximity between cMTs emanating from adjacent nuclei was not observed. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, in most cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forth movements. Only occasionally do all six cMTs orient in one direction, a precondition for long-range nuclear bypassing. Following mitosis, daughter nuclei carry a single SPB with three cMTs. The increased probability that all three cMTs orient in one direction explains the high rate of nuclear bypassing observed in these nuclei. The A. gossypii mitotic spindle was found to be structurally similar to that of Saccharomyces cerevisiae in terms of nuclear microtubule (nMT) number, length distribution and three-dimensional organization even though the two organisms differ significantly in chromosome number. Our results suggest that two nMTs attach to each kinetochore in A. gossypii and not only one nMT like in S. cerevisiae.