Samurai sword sets spindle size.

Although the parts list is nearly complete for many cellular structures, mechanisms that control their size remain poorly understood. Loughlin and colleagues now show that phosphorylation of a single residue of katanin, a microtubule-severing protein, largely accounts for the difference in spindle length between two closely related frogs.

The centrosome protein AKNA regulates neurogenesis via microtubule organization.

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region.

XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215's overall affinity for free tubulin. The mutants' overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a "bonsai" protein, with two TOG domains and a basic region, that has almost full polymerase activity.

Cytoskeletal dynamics in parasites.

Cytoskeletal dynamics are essential for cellular homeostasis and development for both metazoans and protozoans. The function of cytoskeletal elements in protozoans can diverge from that of metazoan cells, with microtubules being more stable and actin filaments being more dynamic. This is particularly striking in protozoan parasites that evolved to enter metazoan cells. Here, we review recent progress towards understanding cytoskeletal dynamics in protozoan parasites, with a focus on divergent properties compared to classic model organisms.

Barrier properties of Nup98 FG phases ruled by FG motif identity and inter-FG spacer length.

Nup98 FG repeat domains comprise hydrophobic FG motifs linked through uncharged spacers. FG motifs capture nuclear transport receptors (NTRs) during nuclear pore complex (NPC) passage, confer inter-repeat cohesion, and condense the domains into a selective phase with NPC-typical barrier properties. We show that shortening inter-FG spacers enhances cohesion, increases phase density, and tightens such barrier - all consistent with a sieve-like phase. Phase separation tolerates mutating the Nup98-typical GLFG motifs, provided domain-hydrophobicity remains preserved. NTR-entry, however, is sensitive to (certain) deviations from canonical FG motifs, suggesting co-evolutionary adaptation. Unexpectedly, we observed that arginines promote FG-phase-entry apparently also by hydrophobic interactions/ hydrogen-bonding and not just through cation-π interactions. Although incompatible with NTR·cargo complexes, a YG phase displays remarkable transport selectivity, particularly for engineered GFPNTR-variants. GLFG to FSFG mutations make the FG phase hypercohesive, precluding NTR-entry. Extending spacers relaxes this hypercohesion. Thus, antagonism between cohesion and NTR·FG interactions is key to transport selectivity.

Calcitic Prisms of The Giant Seashell Pinna Nobilis Form Light Guide Arrays.

The shells of the Pinnidae family are based on a double layer of single-crystal-like calcitic prisms and inner aragonitic nacre, a structure known for its outstanding mechanical performance. However, on the posterior side, shells are missing the nacreous layer, which raises the question of whether there can be any functional role in giving up this mechanical performance. Here, it is demonstrated that the prismatic part of the Pinna nobilis shell exhibits unusual optical properties, whereby each prism acts as an individual optical fiber guiding the ambient light to the inner shell cavity by total internal reflection. This pixelated light channeling enhances both spatial resolution and contrast while reducing angular blurring, an apt combination for acute tracking of a moving object. These findings offer insights into the evolutionary aspects of light-sensing and imaging and demonstrate how an architectured optical system for efficient light-tracking can be based on birefringent ceramics.

Quantitative analysis of conditional gene inactivation using rationally designed, tetracycline-controlled miRNAs.

The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo.

Engineering metaphase spindles: Construction site and building blocks.

In an active, crowded cytoplasm, eukaryotic cells construct metaphase spindles from conserved building blocks to segregate chromosomes. Yet, spindles execute their function in a stunning variety of cell shapes and sizes across orders of magnitude. Thus, the current challenge is to understand how unique mesoscale spindle characteristics emerge from the interaction of molecular collectives. Key components of these collectives are tubulin dimers, which polymerise into microtubules. Despite all conservation, tubulin is a genetically and biochemically complex protein family, and we only begin to uncover how tubulin diversity affects microtubule dynamics and thus spindle assembly. Moreover, it is increasingly appreciated that spindles are dynamically intertwined with the cytoplasm that itself exhibits cell-type specific emergent properties with yet mostly unexplored consequences for spindle construction. Therefore, on our way toward a quantitative picture of spindle function, we need to understand molecular behaviour of the building blocks and connect it to the entire cellular context.

Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors.

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore have the potential to serve as drug targets.1-5 While microtubules and their molecular building block αß-tubulin are established drug targets in a variety of cancers,6,7 we still lack sufficient knowledge of the biochemistry of parasite tubulins to exploit the structural divergence between parasite and human tubulins. For example, it remains to be determined whether compounds of interest can specifically target parasite microtubules without affecting the host cell cytoskeleton. Such mechanistic insights have been limited by the lack of functional parasite tubulin. In this study, we report the purification and characterization of tubulin from Plasmodium falciparum, the causative agent of malaria. We show that the highly purified tubulin is fully functional, as it efficiently assembles into microtubules with specific parameters of dynamic instability. There is a high degree of amino-acid conservation between human and P. falciparum α- and ß-tubulin, sharing approximately 83.7% and 88.5% identity, respectively. However, Plasmodium tubulin is more similar to plant than to mammalian tubulin, raising the possibility of identifying compounds that would selectively disrupt parasite microtubules without affecting the host cell cytoskeleton. As a proof of principle, we describe two compounds that exhibit selective toxicity toward parasite tubulin. Thus, the ability to specifically disrupt protozoan microtubule growth without affecting human microtubules provides an exciting opportunity for the development of novel antimalarials.

Volumetric morphometry reveals spindle width as the best predictor of mammalian spindle scaling.

The function of cellular structures at the mesoscale is dependent on their geometry and proportionality to cell size. The mitotic spindle is a good example why length and shape of intracellular organelles matter. Spindle length determines the distance over which chromosomes will segregate, and spindle shape ensures bipolarity. While we still lack a systematic and quantitative understanding of subcellular morphology, new imaging techniques and volumetric data analysis promise novel insights into scaling relations across different species. Here, we introduce Spindle3D, an open-source plug-in that allows for the quantitative, consistent, and automated analysis of 3D fluorescent data of spindles and chromatin. We systematically analyze different mammalian cell types, including somatic cells, stem cells, and one- and two-cell embryos, to derive volumetric relations of spindle, chromatin, and the cell. Taken together, our data indicate that mitotic spindle width is a robust indicator of spindle volume, which correlates linearly with chromatin and cell volume both within single cell types and across mammalian species.

The Xenopus spindle is as dense as the surrounding cytoplasm.

The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle's material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle's mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle's emergent physical properties-essential to advance predictive frameworks of spindle assembly and function.

Affinity Purification of Label-free Tubulins from Xenopus Egg Extracts.

Cytoplasmic extracts from unfertilized Xenopus eggs have made important contributions to our understanding of microtubule dynamics, spindle assembly, and scaling. Until recently, these in vitro studies relied on the use of heterologous tubulin. This protocol allows for the purification of physiologically relevant Xenopus tubulins in milligram yield, which are a complex mixture of isoforms with various post-translational modifications. The protocol is applicable to any cell or tissue of interest. For complete details on the use and execution of this protocol, please refer to Hirst et al. (2020).

Differences in Intrinsic Tubulin Dynamic Properties Contribute to Spindle Length Control in Xenopus Species.

The function of cellular organelles relates not only to their molecular composition but also to their size. However, how the size of dynamic mesoscale structures is established and maintained remains poorly understood [1-3]. Mitotic spindle length, for example, varies several-fold among cell types and among different organisms [4]. Although most studies on spindle size control focus on changes in proteins that regulate microtubule dynamics [5-8], the contribution of the spindle's main building block, the αß-tubulin heterodimer, has yet to be studied. Apart from microtubule-associated proteins and motors, two factors have been shown to contribute to the heterogeneity of microtubule dynamics: tubulin isoform composition [9, 10] and post-translational modifications [11]. In the past, studying the contribution of tubulin and microtubules to spindle assembly has been limited by the fact that physiologically relevant tubulins were not available. Here, we show that tubulins purified from two closely related frogs, Xenopus laevis and Xenopus tropicalis, have surprisingly different microtubule dynamics in vitro. X. laevis microtubules combine very fast growth and infrequent catastrophes. In contrast, X. tropicalis microtubules grow slower and catastrophe more frequently. We show that spindle length and microtubule mass can be controlled by titrating the ratios of the tubulins from the two frog species. Furthermore, we combine our in vitro reconstitution assay and egg extract experiments with computational modeling to show that differences in intrinsic properties of different tubulins contribute to the control of microtubule mass and therefore set steady-state spindle length.

In Vitro Reconstitution and Imaging of Microtubule Dynamics by Fluorescence and Label-free Microscopy.

Dynamic microtubules are essential for many processes in the lives of eukaryotic cells. To study and understand the mechanisms of microtubule dynamics and regulation, in vitro reconstitution with purified components has proven a vital approach. Imaging microtubule dynamics can be instructive for a given species, isoform composition, or biochemical modification. Here, we describe two methods that visualize microtubule dynamics at high speed and high contrast: (1) total internal reflection fluorescence microscopy and (2) label-free interference reflection microscopy. For complete details on the use and execution of this protocol, please refer to Hirst et al. (2020).

The effects of proliferation status and cell cycle phase on the responses of single cells to chemotherapy.

DNA-damaging chemotherapeutics are widely used in cancer treatments, but for solid tumors they often leave a residual tumor-cell population. Here we investigated how cellular states might affect the response of individual cells in a clonal population to cisplatin, a DNA-damaging chemotherapeutic agent. Using a live-cell reporter of cell cycle phase and long-term imaging, we monitored single-cell proliferation before, at the time of, and after treatment. We found that in response to cisplatin, cells either arrested or died, and the ratio of these outcomes depended on the dose. While we found that the cell cycle phase at the time of cisplatin addition was not predictive of outcome, the proliferative history of the cell was: highly proliferative cells were more likely to arrest than to die, whereas slowly proliferating cells showed a higher probability of death. Information theory analysis revealed that the dose of cisplatin had the greatest influence on the cells' decisions to arrest or die, and that the proliferation status interacted with the cisplatin dose to further guide this decision. These results show an unexpected effect of proliferation status in regulating responses to cisplatin and suggest that slowly proliferating cells within tumors may be acutely vulnerable to chemotherapy.

MTrack: Automated Detection, Tracking, and Analysis of Dynamic Microtubules.

Microtubules are polar, dynamic filaments fundamental to many cellular processes. In vitro reconstitution approaches with purified tubulin are essential to elucidate different aspects of microtubule behavior. To date, deriving data from fluorescence microscopy images by manually creating and analyzing kymographs is still commonplace. Here, we present MTrack, implemented as a plug-in for the open-source platform Fiji, which automatically identifies and tracks dynamic microtubules with sub-pixel resolution using advanced objection recognition. MTrack provides automatic data interpretation yielding relevant parameters of microtubule dynamic instability together with population statistics. The application of our software produces unbiased and comparable quantitative datasets in a fully automated fashion. This helps the experimentalist to achieve higher reproducibility at higher throughput on a user-friendly platform. We use simulated data and real data to benchmark our algorithm and show that it reliably detects, tracks, and analyzes dynamic microtubules and achieves sub-pixel precision even at low signal-to-noise ratios.

Emergent Properties of the Metaphase Spindle.

A metaphase spindle is a complex structure consisting of microtubules and a myriad of different proteins that modulate microtubule dynamics together with chromatin and kinetochores. A decade ago, a full description of spindle formation and function seemed a lofty goal. Here, we describe how work in the last 10 years combining cataloging of spindle components, the characterization of their biochemical activities using single-molecule techniques, and theory have advanced our knowledge. Taken together, these advances suggest that a full understanding of spindle assembly and function may soon be possible.

Intracellular Scaling Mechanisms.

Organelle function is often directly related to organelle size. However, it is not necessarily absolute size but the organelle-to-cell-size ratio that is critical. Larger cells generally have increased metabolic demands, must segregate DNA over larger distances, and require larger cytokinetic rings to divide. Thus, organelles often must scale to the size of the cell. The need for scaling is particularly acute during early development during which cell size can change rapidly. Here, we highlight scaling mechanisms for cellular structures as diverse as centrosomes, nuclei, and the mitotic spindle, and distinguish them from more general mechanisms of size control. In some cases, scaling is a consequence of the underlying mechanism of organelle size control. In others, size-control mechanisms are not obviously related to cell size, implying that scaling results indirectly from cell-size-dependent regulation of size-control mechanisms.

XMAP215 activity sets spindle length by controlling the total mass of spindle microtubules.

Metaphase spindles are microtubule-based structures that use a multitude of proteins to modulate their morphology and function. Today, we understand many details of microtubule assembly, the role of microtubule-associated proteins, and the action of molecular motors. Ultimately, the challenge remains to understand how the collective behaviour of these nanometre-scale processes gives rise to a properly sized spindle on the micrometre scale. By systematically engineering the enzymatic activity of XMAP215, a processive microtubule polymerase, we show that Xenopus laevis spindle length increases linearly with microtubule growth velocity, whereas other parameters of spindle organization, such as microtubule density, lifetime and spindle shape, remain constant. We further show that mass balance can be used to link the global property of spindle size to individual microtubule dynamic parameters. We propose that spindle length is set by a balance of non-uniform nucleation and global microtubule disassembly in a liquid-crystal-like arrangement of microtubules.