Distinct regions of the kinesin-5 C-terminal tail are essential for mitotic spindle midzone localization and sliding force.

Kinesin-5 motor proteins play essential roles during mitosis in most organisms. Their tetrameric structure and plus-end-directed motility allow them to bind to and move along antiparallel microtubules, thereby pushing spindle poles apart to assemble a bipolar spindle. Recent work has shown that the C-terminal tail is particularly important to kinesin-5 function: The tail affects motor domain structure, ATP hydrolysis, motility, clustering, and sliding force measured for purified motors, as well as motility, clustering, and spindle assembly in cells. Because previous work has focused on presence or absence of the entire tail, the functionally important regions of the tail remain to be identified. We have therefore characterized a series of kinesin-5/Cut7 tail truncation alleles in fission yeast. Partial truncation causes mitotic defects and temperature-sensitive growth, while further truncation that removes the conserved BimC motif is lethal. We compared the sliding force generated by cut7 mutants using a kinesin-14 mutant background in which some microtubules detach from the spindle poles and are pushed into the nuclear envelope. These Cut7-driven protrusions decreased as more of the tail was truncated, and the most severe truncations produced no observable protrusions. Our observations suggest that the C-terminal tail of Cut7p contributes to both sliding force and midzone localization. In the context of sequential tail truncation, the BimC motif and adjacent C-terminal amino acids are particularly important for sliding force. In addition, moderate tail truncation increases midzone localization, but further truncation of residues N-terminal to the BimC motif decreases midzone localization.

Quantifying Yeast Microtubules and Spindles Using the Toolkit for Automated Microtubule Tracking (TAMiT).

Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

Distinct regions of the kinesin-5 C-terminal tail are essential for mitotic spindle midzone localization and sliding force.

Kinesin-5 motor proteins play essential roles during mitosis in most organisms. Their tetrameric structure and plus-end-directed motility allow them to bind to and move along antiparallel microtubules, thereby pushing spindle poles apart to assemble a bipolar spindle. Recent work has shown that the C-terminal tail is particularly important to kinesin-5 function: the tail affects motor domain structure, ATP hydrolysis, motility, clustering, and sliding force measured for purified motors, as well as motility, clustering, and spindle assembly in cells. Because previous work has focused on presence or absence of the entire tail, the functionally important regions of the tail remain to be identified. We have therefore characterized a series of kinesin-5/Cut7 tail truncation alleles in fission yeast. Partial truncation causes mitotic defects and temperature-sensitive growth, while further truncation that removes the conserved BimC motif is lethal. We compared the sliding force generated by cut7 mutants using a kinesin-14 mutant background in which some microtubules detach from the spindle poles and are pushed into the nuclear envelope. These Cut7-driven protrusions decreased as more of the tail was truncated, and the most severe truncations produced no observable protrusions. Our observations suggest that the C-terminal tail of Cut7p contributes to both sliding force and midzone localization. In the context of sequential tail truncation, the BimC motif and adjacent C-terminal amino acids are particularly important for sliding force. In addition, moderate tail truncation increases midzone localization, but further truncation of residues N terminal to the BimC motif decreases midzone localization.

Quantifying yeast microtubules and spindles using the Toolkit for Automated Microtubule Tracking (TAMiT).

Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that is solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work, and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae , with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

The kinesin-5 protein Cut7 moves bidirectionally on fission yeast spindles with activity that increases in anaphase.

Kinesin-5 motors are essential to separate mitotic spindle poles and assemble a bipolar spindle in many organisms. These motors crosslink and slide apart antiparallel microtubules via microtubule plus-end-directed motility. However, kinesin-5 localization is enhanced away from antiparallel overlaps. Increasing evidence suggests this localization occurs due to bidirectional motility or trafficking. The purified fission-yeast kinesin-5 protein Cut7 moves bidirectionally, but bidirectionality has not been shown in cells, and the function of the minus-end-directed movement is unknown. Here, we characterized the motility of Cut7 on bipolar and monopolar spindles and observed movement toward both plus- and minus-ends of microtubules. Notably, the activity of the motor increased at anaphase B onset. Perturbations to microtubule dynamics only modestly changed Cut7 movement, whereas Cut7 mutation reduced movement. These results suggest that the directed motility of Cut7 contributes to the movement of the motor. Comparison of the Cut7 mutant and human Eg5 (also known as KIF11) localization suggest a new hypothesis for the function of minus-end-directed motility and spindle-pole localization of kinesin-5s.

Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling.

The essential functions required for mitotic spindle assembly and chromosome biorientation and segregation are not fully understood, despite extensive study. To illuminate the combinations of ingredients most important to align and segregate chromosomes and simultaneously assemble a bipolar spindle, we developed a computational model of fission-yeast mitosis. Robust chromosome biorientation requires progressive restriction of attachment geometry, destabilization of misaligned attachments, and attachment force dependence. Large spindle length fluctuations can occur when the kinetochore-microtubule attachment lifetime is long. The primary spindle force generators are kinesin-5 motors and crosslinkers in early mitosis, while interkinetochore stretch becomes important after biorientation. The same mechanisms that contribute to persistent biorientation lead to segregation of chromosomes to the poles after anaphase onset. This model therefore provides a framework to interrogate key requirements for robust chromosome biorientation, spindle length regulation, and force generation in the spindle.

Before a cell divides, it must make a copy of its genetic material and then promptly split in two. This process, called mitosis, is coordinated by many different molecular machines. The DNA is copied, then the duplicated chromosomes line up at the middle of the cell. Next, an apparatus called the mitotic spindle latches onto the chromosomes before pulling them apart. The mitotic spindle is a bundle of long, thin filaments called microtubules. It attaches to chromosomes at the kinetochore, the point where two copied chromosomes are cinched together in their middle. Proper cell division is vital for the healthy growth of all organisms, big and small, and yet some parts of the process remain poorly understood despite extensive study. Specifically, there is more to learn about how the mitotic spindle self-assembles, and how microtubules and kinetochores work together to correctly orient and segregate chromosomes into two sister cells. These nanoscale processes are happening a hundred times a minute, so computer simulations are a good way to test what we know. Edelmaier et al. developed a computer model to simulate cell division in fission yeast, a species of yeast often used to study fundamental processes in the cell. The model simulates how the mitotic spindle assembles, how its microtubules attach to the kinetochore and the force required to pull two sister chromosomes apart. Building the simulation involved modelling interactions between the mitotic spindle and kinetochore, their movement and forces applied. To test its accuracy, model simulations were compared to recordings of the mitotic spindle ­ including its length, structure and position ­ imaged from dividing yeast cells. Running the simulation, Edelmaier et al. found that several key effects are essential for the proper movement of chromosomes in mitosis. This includes holding chromosomes in the correct orientation as the mitotic spindle assembles and controlling the relative position of microtubules as they attach to the kinetochore. Misaligned attachments must also be readily deconstructed and corrected to prevent any errors. The simulations also showed that kinetochores must begin to exert more force (to separate the chromosomes) once the mitotic spindle is attached correctly. Altogether, these findings improve the current understanding of how the mitotic spindle and its counterparts control cell division. Errors in chromosome segregation are associated with birth defects and cancer in humans, and this new simulation could potentially now be used to help make predictions about how to correct mistakes in the process.

3D electron tomographic and biochemical analysis of ER, Golgi and trans Golgi network membrane systems in stimulated Venus flytrap (Dionaea muscipula) glandular cells.

BACKGROUND: The insect-trapping leaves of Dionaea muscipula provide a model for studying the secretory pathway of an inducible plant secretory system. The leaf glands were induced with bovine serum albumin to secrete proteases that were characterized via zymogram activity gels over a 6-day period. The accompanying morphological changes of the endoplasmic reticulum (ER) and Golgi were analyzed using 3D electron tomography of glands preserved by high-pressure freezing/freeze substitution methods. RESULTS: Secretion of multiple cysteine and aspartic proteases occurred biphasically. The majority of the Golgi was organized in clusters consisting of 3-6 stacks surrounded by a cage-like system of ER cisternae. In these clusters, all Golgi stacks were oriented with their cis-most C1 cisterna facing an ER export site. The C1 Golgi cisternae varied in size and shape consistent with the hypothesis that they form de novo. Following induction, the number of ER-bound polysomes doubled, but no increase in COPII vesicles was observed. Golgi changes included a reduction in the number of cisternae per stack and a doubling of cisternal volume without increased surface area. Polysaccharide molecules that form the sticky slime cause swelling of the trans and trans Golgi network (TGN) cisternae. Peeling of the trans-most cisternae gives rise to free TGN cisternae. One day after gland stimulation, the free TGNs were frequently associated with loose groups of oriented actin-like filaments which were not seen in any other samples. CONCLUSIONS: These findings suggest that the secretory apparatus of resting gland cells is "overbuilt" to enable the cells to rapidly up-regulate lytic enzyme production and secretion in response to prey trapping.

Physical determinants of bipolar mitotic spindle assembly and stability in fission yeast.

Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition; this is the most fundamental construction problem that the spindle must solve. Microtubules, motors, and cross-linkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown. We describe a physical model that exhibits de novo bipolar spindle formation. We began with physical properties of fission-yeast spindle pole body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive cross-linkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective self-assembly. By varying the features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. When kinesin-5 motors are present, their bidirectionality is essential, but spindles can form in the presence of passive cross-linkers alone. We also identify characteristic failed states of spindle assembly-the persistent monopole, X spindle, separated asters, and short spindle, which are avoided by the creation and maintenance of antiparallel microtubule overlaps. Our model can guide the identification of new, multifaceted strategies to induce mitotic catastrophes; these would constitute novel strategies for cancer chemotherapy.

Contributions of Microtubule Dynamic Instability and Rotational Diffusion to Kinetochore Capture.

Microtubule dynamic instability allows search and capture of kinetochores during spindle formation, an important process for accurate chromosome segregation during cell division. Recent work has found that microtubule rotational diffusion about minus-end attachment points contributes to kinetochore capture in fission yeast, but the relative contributions of dynamic instability and rotational diffusion are not well understood. We have developed a biophysical model of kinetochore capture in small fission-yeast nuclei using hybrid Brownian dynamics/kinetic Monte Carlo simulation techniques. With this model, we have studied the importance of dynamic instability and microtubule rotational diffusion for kinetochore capture, both to the lateral surface of a microtubule and at or near its end. Over a range of biologically relevant parameters, microtubule rotational diffusion decreased capture time, but made a relatively small contribution compared to dynamic instability. At most, rotational diffusion reduced capture time by 25%. Our results suggest that while microtubule rotational diffusion can speed up kinetochore capture, it is unlikely to be the dominant physical mechanism for typical conditions in fission yeast. In addition, we found that when microtubules undergo dynamic instability, lateral captures predominate even in the absence of rotational diffusion. Counterintuitively, adding rotational diffusion to a dynamic microtubule increases the probability of end-on capture.

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast.

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8-deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8-induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen.

Cis-Golgi cisternal assembly and biosynthetic activation occur sequentially in plants and algae.

The cisternal progression/maturation model of Golgi trafficking predicts that cis-Golgi cisternae are formed de novo on the cis-side of the Golgi. Here we describe structural and functional intermediates of the cis cisterna assembly process in high-pressure frozen algae (Scherffelia dubia, Chlamydomonas reinhardtii) and plants (Arabidopsis thaliana, Dionaea muscipula; Venus flytrap) as determined by electron microscopy, electron tomography and immuno-electron microscopy techniques. Our findings are as follows: (i) The cis-most (C1) Golgi cisternae are generated de novo from cisterna initiators produced by the fusion of 3-5 COPII vesicles in contact with a C2 cis cisterna. (ii) COPII vesicles fuel the growth of the initiators, which then merge into a coherent C1 cisterna. (iii) When a C1 cisterna nucleates its first cisterna initiator it becomes a C2 cisterna. (iv) C2-Cn cis cisternae grow through COPII vesicle fusion. (v) ER-resident proteins are recycled from cis cisternae to the ER via COPIa-type vesicles. (vi) In S. dubia the C2 cisternae are capable of mediating the self-assembly of scale protein complexes. (vii) In plants, ∼90% of native α-mannosidase I localizes to medial Golgi cisternae. (viii) Biochemical activation of cis cisternae appears to coincide with their conversion to medial cisternae via recycling of medial cisterna enzymes. We propose how the different cis cisterna assembly intermediates of plants and algae may actually be related to those present in the ERGIC and in the pre-cis Golgi cisterna layer in mammalian cells.