Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes.

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.

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.

Regulation of microtubule dynamics, mechanics and function through the growing tip.

Microtubule dynamics and their control are essential for the normal function and division of all eukaryotic cells. This plethora of functions is, in large part, supported by dynamic microtubule tips, which can bind to various intracellular targets, generate mechanical forces and couple with actin microfilaments. Here, we review progress in the understanding of microtubule assembly and dynamics, focusing on new information about the structure of microtubule tips. First, we discuss evidence for the widely accepted GTP cap model of microtubule dynamics. Next, we address microtubule dynamic instability in the context of structural information about assembly intermediates at microtubule tips. Three currently discussed models of microtubule assembly and dynamics are reviewed. These are considered in the context of established facts and recent data, which suggest that some long-held views must be re-evaluated. Finally, we review structural observations about the tips of microtubules in cells and describe their implications for understanding the mechanisms of microtubule regulation by associated proteins, by mechanical forces and by microtubule-targeting drugs, prominently including cancer chemotherapeutics.

Correction: Brownian dynamics simulation of protofilament relaxation during rapid freezing.

[This corrects the article DOI: 10.1371/journal.pone.0247022.].

Anaphase A.

Anaphase A is the motion of recently separated chromosomes to the spindle pole they face. It is accompanied by the shortening of kinetochore-attached microtubules. The requisite tubulin depolymerization may occur at kinetochores, at poles, or both, depending on the species and/or the time in mitosis. These depolymerization events are local and suggest that cells regulate microtubule dynamics in specific places, presumably by the localization of relevant enzymes and microtubule-associated proteins to specific loci, such as pericentriolar material and outer kinetochores. Motor enzymes can contribute to anaphase A, both by altering microtubule stability and by pushing or pulling microtubules through the cell. The generation of force on chromosomes requires couplings that can both withstand the considerable force that spindles can generate and simultaneously permit tubulin addition and loss. This chapter reviews literature on the molecules that regulate anaphase microtubule dynamics, couple dynamic microtubules to kinetochores and poles, and generate forces for microtubule and chromosome motion.

Brownian dynamics simulation of protofilament relaxation during rapid freezing.

Electron cryo-microscopy (Cryo-EM) is a powerful method for visualizing biological objects with up to near-angstrom resolution. Instead of chemical fixation, the method relies on very rapid freezing to immobilize the sample. Under these conditions, crystalline ice does not have time to form and distort structure. For many practical applications, the rate of cooling is fast enough to consider sample immobilization instantaneous, but in some cases, a more rigorous analysis of structure relaxation during freezing could be essential. This difficult yet important problem has been significantly under-reported in the literature, despite spectacular recent developments in Cryo-EM. Here we use Brownian dynamics modeling to examine theoretically the possible effects of cryo-immobilization on the apparent shapes of biological polymers. The main focus of our study is on tubulin protofilaments. These structures are integral parts of microtubules, which in turn are key elements of the cellular skeleton, essential for intracellular transport, maintenance of cell shape, cell division and migration. We theoretically examine the extent of protofilament relaxation within the freezing time as a function of the cooling rate, the filament's flexural rigidity, and the effect of cooling on water's viscosity. Our modeling suggests that practically achievable cooling rates are not rapid enough to capture tubulin protofilaments in conformations that are incompletely relaxed, suggesting that structures seen by cryo-EM are good approximations to physiological shapes. This prediction is confirmed by our analysis of curvatures of tubulin protofilaments, using samples, prepared and visualized with a variety of methods. We find, however, that cryofixation may capture incompletely relaxed shapes of more flexible polymers, and it may affect Cryo-EM-based measurements of their persistence lengths. This analysis will be valuable for understanding of structures of different types of biopolymers, observed with Cryo-EM.

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.

Electron tomography reveals aspects of spindle structure important for mechanical stability at metaphase.

Metaphase spindles exert pole-directed forces on still-connected sister kinetochores. The spindle must counter these forces with extensive forces to prevent spindle collapse. In small spindles, kinetochore microtubules (KMTs) connect directly with the poles, and countering forces are supplied either by interdigitating MTs that form interpolar bundles or by astral MTs connected to the cell cortex. In bigger spindles, particularly those without structured poles, the origin of extensive forces is less obvious. We have used electron tomography of well-preserved metaphase cells to obtain structural evidence about interactions among different classes of MTs in metaphase spindles from Chlamydomonas rheinhardti and two strains of cultured mammalian cells. In all these spindles, KMTs approach close to and cross-bridge with the minus ends of non-KMTs, which form a framework that interdigitates near the spindle equator. Although this structure is not pole-connected, its organization suggests that it can support kinetochore tension. Analogous arrangements of MTs have been seen in even bigger spindles, such as metaphase spindles in Haemanthus endosperm and frog egg extracts. We present and discuss a hypothesis that rationalizes changes in spindle design with spindle size based on the negative exponential distribution of MT lengths in dynamically unstable populations of tubulin polymers.

Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments.

We used electron tomography to examine microtubules (MTs) growing from pure tubulin in vitro as well as two classes of MTs growing in cells from six species. The tips of all these growing MTs display bent protofilaments (PFs) that curve away from the MT axis, in contrast with previously reported MTs growing in vitro whose tips are either blunt or sheetlike. Neither high pressure nor freezing is responsible for the PF curvatures we see. The curvatures of PFs on growing and shortening MTs are similar; all are most curved at their tips, suggesting that guanosine triphosphate-tubulin in solution is bent and must straighten to be incorporated into the MT wall. Variations in curvature suggest that PFs are flexible in their plane of bending but rigid to bending out of that plane. Modeling by Brownian dynamics suggests that PF straightening for MT growth can be achieved by thermal motions, providing a simple mechanism with which to understand tubulin polymerization.

Assessing the Contributions of Motor Enzymes and Microtubule Dynamics to Mitotic Chromosome Motions.

During my graduate work with Keith Porter, I became fascinated by the mitotic spindle, an interest that has motivated much of my scientific work ever since. I began spindle studies by using electron microscopes, instruments that have made significant contributions to our understanding of spindle organization. Such instruments have helped to elucidate the distributions of spindle microtubules, the interactions among them, their molecular polarity, and their associations with both kinetochores and spindle poles. Our lab has also investigated some processes of spindle physiology: microtubule dynamics, the actions of microtubule-associated proteins (including motor enzymes), the character of forces generated by specific spindle components, and factors that control mitotic progression. Here, I give a personal perspective on some of this intellectual history and on what recent discoveries imply about the mechanisms of chromosome motion.

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.

Preparing Fission Yeast for Electron Microscopy.

Freezing samples while simultaneously subjecting them to a rapid increase in pressure, which inhibits ice crystal formation, is a reliable method for cryofixing fission yeast. The procedure consists simply of harvesting cells and loading them into a high-pressure freezer (HPF), and then operating the device. If equipment for high-pressure freezing is not available, fission yeast can be frozen by plunging a monolayer of cells into a liquid cryogen, usually ethane or propane. Unlike the HPF, where relatively large volumes of cells can be frozen in a single run, plunge freezing requires cells to be dispersed in a layer <20 µm thick. Unless frozen cells are to be imaged in the vitreous state, they must be fixed, dehydrated, and embedded for subsequent study by transmission electron microscopy; warming frozen cells without fixation badly damages cell structure. Fixation is best accomplished by freeze-substitution, a process in which frozen water is removed from samples by a water-miscible solvent that is liquid at a temperature low enough to prevent the cellular water from recrystallizing. Low concentrations of chemical fixatives and stains are generally added to this solvent such that they permeate the cells as the water is replaced. The activity of these additives is quite limited at the low temperatures required for minimizing ice crystal formation, but they are in the right place to react effectively as the cells warm up. Step-by-step protocols for HPF, plunge freezing, and freeze-substitution are provided here.

Immunolocalization of Proteins in Fission Yeast by Electron Microscopy.

Electron microscopy (EM) immunolocalization of antigens in fission yeast can be accomplished with cells processed by rapid freezing and freeze-substitution followed by embedding in acrylic or methacrylate resins. Microtome sections of embedded cells are collected onto EM grids. Primary antibodies to the antigen of interest, followed by secondary antibodies conjugated to colloidal gold, are allowed to bind to antigens at the surface of these plastic sections. This type of postembed labeling provides information on antigen localization to a resolution of 10-20 nm, depending on the size of the metal particle used, the form of the antibody (Fab vs. complete IgG or IgM), and whether direct or indirect labeling is used. The method has the potential to map macromolecules in three dimensions in a relatively large volume when thin (30-60-nm) serial sections are labeled, imaged, aligned, and modeled to create a representative volume. The biggest challenge of this technique is the necessary compromise between the preservation of cellular ultrastructure and the preservation of antigen reactivity. The protocols described here show how to immunolabel samples for EM and include suggestions for overcoming challenges related to antigen preservation.

Cryoelectron Microscopy of Fission Yeast.

Fission yeast cells can be prepared for electron microscopy (EM) in the frozen-hydrated state. This eliminates the requirement for dehydration and heavy metal staining when preparing samples for EM. As with room temperature imaging, however, the yeast must be sectioned to make them thin enough for transmission of the electron beam. Cutting sections of vitreous ice with a microtome is challenging. An alternative method that uses a focused ion beam to make a thin sample by milling away much of the sample at liquid nitrogen temperatures is under development but is not yet available for routine use. Imaging frozen-hydrated samples by EM is also a challenge. The technique involves battling low image contrast, high sensitivity to the electron beam, and mechanical distortions produced during the sectioning process. When used successfully, however, the method holds promise of providing excellent molecular detail without the disruption characteristic of dehydration or isolating a structure from its cellular environment. Cryo-EM of tilted views can be used to examine small structures and macromolecular complexes in their native cellular environment. If a structure exists in multiple copies, or has a repeating unit, it can be investigated at higher resolution using subvolume averaging. This protocol focuses on the preparation of cells for cryo-EM.

Electron Microscopy of Fission Yeast.

Electron microscopy (EM) can provide images of cells with a spatial resolution that significantly surpasses that available from light microscopy (LM), even with modern methods that give LM "super resolution." However, EM resolution comes with costs in time spent with sample preparation, expense of instrumentation, and concerns regarding sample preparation artifacts. It is therefore important to know the limitations of EM as well as its strengths. Here we describe the most reliable methods for the preservation of fission yeast cells currently available. We describe the properties of images obtained by transmission EM (TEM) and contrast them with images from scanning EM (SEM). We also show how one can make three-dimensional TEM images and discuss several approaches to address the problem of localizing specific proteins within cells. We give references to work by others who have pursued similar goals with different methods, and we discuss briefly the complex subject of image interpretation.

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.

Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium "Candidatus Pelagibacter ubique".

SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study "Candidatus Pelagibacter ubique" strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. Frozen-hydrated cells, which were preserved in a life-like state, had an average cell volume (enclosed by the outer membrane) of 0.037 ± 0.011 µm3 Strikingly, the periplasmic space occupied ∼20% to 50% of the total cell volume in log-phase cells and ∼50% to 70% in stationary-phase cells. The nucleoid occupied the convex side of the crescent-shaped cells and the ribosomes predominantly occupied the concave side, at a relatively high concentration of 10,000 to 12,000 ribosomes/µm3 Outer membrane pore complexes, likely composed of PilQ, were frequently observed in both log-phase and stationary-phase cells. Long filaments, most likely type IV pili, were found on dividing cells. The physical dimensions, intracellular organization, and morphological changes throughout the life cycle of "Ca. Pelagibacter ubique" provide structural insights into the functional adaptions of these oligotrophic ultramicrobacteria to their habitat. IMPORTANCE: Bacterioplankton of the SAR11 clade (Pelagibacterales) are of interest because of their global biogeochemical significance and because they appear to have been molded by unusual evolutionary circumstances that favor simplicity and efficiency. They have adapted to an ecosystem in which nutrient concentrations are near the extreme limits at which transport systems can function adequately, and they have evolved streamlined genomes to execute only functions essential for life. However, little is known about the actual size limitations and cellular features of living oligotrophic ultramicrobacteria. In this study, we have used cryo-electron tomography to obtain accurate physical information about the cellular architecture of "Candidatus Pelagibacter ubique," the first cultivated member of the SAR11 clade. These results provide foundational information for answering questions about the cell architecture and functions of these ultrasmall oligotrophic bacteria.

A Brief History of Research on Mitotic Mechanisms.

This chapter describes in summary form some of the most important research on chromosome segregation, from the discovery and naming of mitosis in the nineteenth century until around 1990. It gives both historical and scientific background for the nine chapters that follow, each of which provides an up-to-date review of a specific aspect of mitotic mechanism. Here, we trace the fruits of each new technology that allowed a deeper understanding of mitosis and its underlying mechanisms. We describe how light microscopy, including phase, polarization, and fluorescence optics, provided descriptive information about mitotic events and also enabled important experimentation on mitotic functions, such as the dynamics of spindle fibers and the forces generated for chromosome movement. We describe studies by electron microscopy, including quantitative work with serial section reconstructions. We review early results from spindle biochemistry and genetics, coupled to molecular biology, as these methods allowed scholars to identify key molecular components of mitotic mechanisms. We also review hypotheses about mitotic mechanisms whose testing led to a deeper understanding of this fundamental biological event. Our goal is to provide modern scientists with an appreciation of the work that has laid the foundations for their current work and interests.

Mitosis.

SUMMARYAll eukaryotic cells prepare for cell division by forming a "mitotic spindle"-a bipolar machine made from microtubules (MTs) and many associated proteins. This device organizes the already duplicated DNA so one copy of each chromosome attaches to each end of the spindle. Both formation and function of the spindle require controlled MT dynamics, as well as the actions of multiple motor enzymes. Spindle-driven motions separate the duplicated chromosomes into two distinct sets that are then moved toward opposite ends of the cell. The two cells that subsequently form by cytokinesis, therefore, contain all the genes needed to grow and divide again.