Human dynein-dynactin is a fast processive motor in living cells.

Minus-end directed transport along microtubules in eukaryotes is primarily mediated by cytoplasmic dynein and its cofactor dynactin. Significant advances have been made in recent years characterizing human dynein-dynactin structure and function using in vitro assays, however, there is limited knowledge about the motile properties and functional organization of dynein-dynactin in living human cells. Total internal reflection fluorescence microscopy (TIRFM) of CRISPR-engineered human cells is employed here to visualize fluorescently tagged dynein heavy chain (DHC) and p50 with high spatio-temporal resolution. We find that p50 and DHC exhibit indistinguishable motility properties in their velocities, run lengths, and run times. The dynein-dynactin complexes are fast (∼1.2 µm/s) and typically run for several microns (∼2.7 µm). Quantification of the fluorescence intensities of motile puncta reveals that dynein-dynactin runs are mediated by at least one DHC dimer while the velocity is consistent with that measured for double dynein (two DHC dimers) complexes in vitro.

Lateral and longitudinal compaction of PRC1 overlap zones drives stabilization of interzonal microtubules.

During anaphase, antiparallel-overlapping midzone microtubules elongate and form bundles, contributing to chromosome segregation and the location of contractile ring formation. Midzone microtubules are dynamic in early but not late anaphase; however, the kinetics and mechanisms of stabilization are incompletely understood. Using photoactivation of cells expressing PA-EGFP-α-tubulin we find that immediately after anaphase onset, a single highly dynamic population of midzone microtubules is present; as anaphase progresses, both dynamic and stable populations of midzone microtubules coexist. By mid-cytokinesis, only static, non-dynamic microtubules are detected. The velocity of microtubule sliding also decreases as anaphase progresses, becoming undetectable by late anaphase. Following depletion of PRC1, midzone microtubules remain highly dynamic in anaphase and fail to form static arrays in telophase despite furrowing. Cells depleted of Kif4a contain elongated PRC1 overlap zones and fail to form static arrays in telophase. Cells blocked in cytokinesis form short PRC1 overlap zones that do not coalesce laterally; these cells also fail to form static arrays in telophase. Together, our results demonstrate that dynamic turnover and sliding of midzone microtubules is gradually reduced during anaphase and that the final transition to a static array in telophase requires both lateral and longitudinal compaction of PRC1 containing overlap zones.

Measurement of Microtubule Stability in Mammalian Cells.

The microtubule cytoskeleton is essential for various biological processes such as the intracellular distribution of molecules and organelles, cell morphogenesis, chromosome segregation, and specification of the location of contractile ring formation. Distinct cell types contain microtubules with different extents of stability. For example, microtubules in neurons are highly stabilized to support organelle (or vesicular) transport over large distances, and microtubules in motile cells are more dynamic. In some cases, such as the mitotic spindle, both dynamic and stable microtubules coexist. Alteration of microtubule stability is connected to disease states, making understanding microtubule stability an important area of research. Methods to measure microtubule stability in mammalian cells are described here. Together, these approaches allow microtubule stability to be measured qualitatively or semiquantitatively following staining for post-translational modifications of tubulin or treating cells with microtubule destabilizing agents such as nocodazole. Microtubule stability can also be measured quantitatively by performing fluorescence recovery after photobleaching or fluorescence photoactivation of tubulin in live cells. These methods should be helpful for those seeking to understand microtubule dynamics and stabilization. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Fixing and staining cells for tubulin post-translational modifications Basic Protocol 2: Evaluating microtubule stability following treatment with nocodazole in live or fixed cells Basic Protocol 3: Measurement of microtubule dynamic turnover by quantification of fluorescence recovery after photobleaching Basic Protocol 4: Measurement of microtubule dynamic turnover by quantification of dissipation of fluorescence after photoactivation.

Naegleria's mitotic spindles are built from unique tubulins and highlight core spindle features.

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis and thus represent a unique system for studying the evolution and functional specificity of mitotic tubulins and the spindles they assemble. Previous studies show that Naegleria amoebae express a divergent α-tubulin during mitosis, and we now show that Naegleria amoebae express a second mitotic α- and two mitotic ß-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and ß-tubulins and contain residues that suggest distinct microtubule properties. These distinct residues are conserved in mitotic tubulin homologs of the "brain-eating amoeba" Naegleria fowleri, making them potential drug targets. Using quantitative light microscopy, we find that Naegleria's mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria's spindle is twisted, and its length increases during mitosis, suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers and secondary bundles function as bridging fibers.

A OneStep Solution to Fix and Stain Cells for Correlative Live and Fixed Microscopy.

Correlating the location of subcellular structures with dynamic cellular behaviors is difficult when working with organisms that lack the molecular genetic tools needed for expressing fluorescent protein fusions. Here, we describe a protocol for fixing, permeabilizing, and staining cells in a single step while imaging on a microscope. In contrast to traditional, multi-step fixing and staining protocols that take hours, the protocol outlined here achieves satisfactory staining within minutes. This approach takes advantage of well-characterized small molecules that stain specific subcellular structures, including nuclei, mitochondria, and actin networks. Direct visualization of the entire process allows for rapid optimization of cell fixation and staining, as well as straightforward identification of fixation artifacts. Moreover, live imaging prior to fixation reveals the dynamic history of cellular features, making it particularly useful for model systems without the capacity for expressing fluorescent protein fusions. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Fixing, permeabilizing, and staining mammalian cells in one step on the microscope.

The multifunctional spindle midzone in vertebrate cells at a glance.

During anaphase, a microtubule-containing structure called the midzone forms between the segregating chromosomes. The midzone is composed of an antiparallel array of microtubules and numerous microtubule-associated proteins that contribute to midzone formation and function. In many cells, the midzone is an important source of signals that specify the location of contractile ring assembly and constriction. The midzone also contributes to the events of anaphase by generating forces that impact chromosome segregation and spindle elongation; some midzone components contribute to both processes. The results of recent experiments have increased our understanding of the importance of the midzone, a microtubule array that has often been overlooked. This Journal of Cell Science at a Glance article will review, and illustrate on the accompanying poster, the organization, formation and dynamics of the midzone, and discuss open questions for future research.

Kinesin-5 Regulation and Function in Mitosis.

Chromosome segregation during cell division requires a bipolar mitotic spindle. Therefore, how the spindle is formed, maintained, and functions is of fundamental importance for all eukaryotic cells. Members of the evolutionarily conserved kinesin-5 family of motor proteins have been shown to play an essential role in spindle formation by generating forces that establish and maintain spindle bipolarity and contribute to spindle elongation. Recent work demonstrates that accessory proteins and post-translational modifications regulate the localization and activity of kinesin-5 motors in cells. In addition, some kinesin-5 motors can move toward the microtubule plus-or-minus end. This new information provides insight into how these motors function during mitosis.

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS.

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed 'early risers'. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5.

Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin-5, Eg5, which generates outward force to establish spindle bipolarity. Our results demonstrate that Eg5 is phosphorylated in cultured cells by Src family kinases (SFKs) at three sites in the motor head: Y125, Y211, and Y231. Mutation of these sites diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Y211 in LLC-Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the nonphosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. We propose that this phosphoregulatory mechanism tunes Eg5 enzymatic activity for optimal spindle morphology.

Regulation of Kif15 localization and motility by the C-terminus of TPX2 and microtubule dynamics.

Mitotic motor proteins generate force to establish and maintain spindle bipolarity, but how they are temporally and spatially regulated in vivo is unclear. Prior work demonstrated that a microtubule-associated protein, TPX2, targets kinesin-5 and kinesin-12 motors to spindle microtubules. The C-terminal domain of TPX2 contributes to the localization and motility of the kinesin-5, Eg5, but it is not known whether this domain regulates kinesin-12, Kif15. We found that the C-terminal domain of TPX2 contributes to the localization of Kif15 to spindle microtubules in cells and suppresses motor walking in vitro. Kif15 and Eg5 are partially redundant motors, and overexpressed Kif15 can drive spindle formation in the absence of Eg5 activity. Kif15-dependent bipolar spindle formation in vivo requires the C-terminal domain of TPX2. In the spindle, fluorescent puncta of GFP-Kif15 move toward the equatorial region at a rate equivalent to microtubule growth. Reduction of microtubule growth with paclitaxel suppresses GFP-Kif15 motility, demonstrating that dynamic microtubules contribute to Kif15 behavior. Our results show that the C-terminal region of TPX2 regulates Kif15 in vitro, contributes to motor localization in cells, and is required for Kif15 force generation in vivo and further reveal that dynamic microtubules contribute to Kif15 behavior in vivo.

Using Fluorescence Microscopy to Study Mitosis.

Fluorescence microscopy is one of the most important approaches in the cell biologist's toolbox for studying the mitotic spindle. In fact, many of the key insights into our understanding of mitosis have been enabled by the visualization of mitotic processes using fluorescence microscopy. Here, we summarize some of the important considerations for imaging mitosis using fluorescence microscopy. Because light can damage live cells, we emphasize the importance of minimizing cellular damage while obtaining informative images.

TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule.

The microtubule-associated protein, TPX2, regulates the activity of the mitotic kinesin, Eg5, but the mechanism of regulation is not established. Using total internal reflection fluorescence microscopy, we observed that Eg5, in extracts of mammalian cells expressing Eg5-EGFP, moved processively toward the microtubule plus-end at an average velocity of 14 nm/s. TPX2 bound to microtubules with an apparent dissociation constant of ∼ 200 nm, and microtubule binding was not dependent on the C-terminal tails of tubulin. Using single molecule assays, we found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration. To determine the region(s) of Eg5 that is required for interaction with TPX2, we performed microtubule gliding assays. Dimeric, but not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays.

Eg5 restricts anaphase B spindle elongation in mammalian cells.

During anaphase, overlapping antiparallel microtubules in the spindle interzone elongate and contribute to chromosome segregation. Kinesin-5 family members are required for spindle elongation in some cells, but in other cases they restrict elongation acting like a brake. To determine how kinesin-5 contributes to spindle elongation in mammalian cells, we treated LLC-Pk1 epithelial cells with small molecule inhibitors of the mammalian kinesin-5, Eg5, at anaphase onset and measured the rate and extent of spindle pole separation using multidimensional tracking of centrosomes in cells expressing GFP-γ-tubulin. Centrosome separation was biphasic, with an initial fast phase followed by a slower phase. Treatment with the small molecule inhibitor, STLC, which weakens the interaction of Eg5 with microtubules, resulted in an increase in the rate of centrosome separation. Conversely, treatment with FCPT, which induces a rigor-like interaction of Eg5 with microtubules, reduced the rate of spindle elongation. In control cells, GFP-Eg5 was localized to spindle microtubules and accumulated in the interzone as anaphase progressed. Spindle fluorescence of GFP-Eg5 was decreased following treatment with STLC and increased in cells treated with FCPT. In anaphase cells, cortical dynein increases and rocking motion of spindle poles was detected consistent with the possibility that dynein mediates spindle elongation. In summary, our results demonstrate that Eg5 is not required for spindle elongation, and in fact, restricts the rate of spindle elongation in mammalian cells.

Microtubule motors: doin' it without dynactin.

The minus-end directed microtubule motor protein cytoplasmic dynein contributes to many aspects of cell division and it is generally believed that these mitotic functions require the dynein activator and processivity factor, dynactin. New research now shows that dynein accomplishes many of its mitotic functions without dynactin.

Cell cycle-regulated cortical dynein/dynactin promotes symmetric cell division by differential pole motion in anaphase.

In cultured mammalian cells, how dynein/dynactin contributes to spindle positioning is poorly understood. To assess the role of cortical dynein/dynactin in this process, we generated mammalian cell lines expressing localization and affinity purification (LAP)-tagged dynein/dynactin subunits from bacterial artificial chromosomes and observed asymmetric cortical localization of dynein and dynactin during mitosis. In cells with asymmetrically positioned spindles, dynein and dynactin were both enriched at the cortex distal to the spindle. NuMA, an upstream targeting factor, localized asymmetrically along the cell cortex in a manner similar to dynein and dynactin. During spindle motion toward the distal cortex, dynein and dynactin were locally diminished and subsequently enriched at the new distal cortex. At anaphase onset, we observed a transient increase in cortical dynein, followed by a reduction in telophase. Spindle motion frequently resulted in cells entering anaphase with an asymmetrically positioned spindle. These cells gave rise to symmetric daughter cells by dynein-dependent differential spindle pole motion in anaphase. Our results demonstrate that cortical dynein and dynactin dynamically associate with the cell cortex in a cell cycle-regulated manner and are required to correct spindle mispositioning in LLC-Pk1 epithelial cells.

Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2.

Kinesin-5 is an essential mitotic motor. However, how its spatial-temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification-tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end-directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end-directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2.

Using cultured mammalian cells to study mitosis.

Diverse cell types have been used to study various aspects of mitosis. Early investigators focused primarily on cells that were suited to morphological studies. More recently, experimental systems have been developed to study both morphology and the molecular basis of chromosome motion and cell-cycle regulation. This article briefly reviews cell types that have been used to study mitosis in live cells. It then discusses cell lines that have been used to examine mitosis in cultured mammalian cells and summarizes the methods that are used to culture and study these cells.