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.

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.

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.

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.

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. While microtubules and their molecular building block αβ-tubulin are established drug targets in a variety of cancers, 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.

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.

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.

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, reconstitution with purified components has proven a vital approach. Imaging microtubule dynamics can be instructive for a given species, isoform composition, or biochemical modification.

Affinity Purification of Label-free Tubulins from Egg Extracts.

Cytoplasmic extracts from unfertilized eggs have made important contributions to our understanding of microtubule dynamics, spindle assembly, and scaling. Until recently, these studies relied on the use of heterologous tubulin. This protocol allows for the purification of physiologically relevant tubulins in milligram yield, which are a complex mixture of isoforms with various post-translational modifications.

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].

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.

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.

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 progenitors that delaminate and settle in the subventricular zone in enlarged brain regions. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination.

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.

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.

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.

GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration.

One-step purification of assembly-competent tubulin from diverse eukaryotic sources.

We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions.

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.

Isolation of centrosomes from cultured cells.

The centrosome is a unique organelle that nucleates and organizes the interphase microtubule array and facilitates bipolar spindle assembly during mitosis. Isolated centrosomes are ideal starting materials for biochemical and structural studies as well as for functional assays to study microtubule-dependent processes. In fact, they are hitherto the only system to study radial microtubule arrays in vitro.

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.

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.

CaM kinase II initiates meiotic spindle depolymerization independently of APC/C activation.

Altered spindle microtubule dynamics at anaphase onset are the basis for chromosome segregation. In Xenopus laevis egg extracts, increasing free calcium levels and subsequently rising calcium-calmodulin-dependent kinase II (CaMKII) activity promote a release from meiosis II arrest and reentry into anaphase. CaMKII induces the activation of the anaphase-promoting complex/cyclosome (APC/C), which destines securin and cyclin B for degradation to allow chromosome separation and mitotic exit.

EML3 is a nuclear microtubule-binding protein required for the correct alignment of chromosomes in metaphase.

Assembly of the mitotic spindle requires a global change in the activity and constitution of the microtubule-binding-protein array at mitotic onset. An important subset of mitotic microtubule-binding proteins localises to the nucleus in interphase and essentially contributes to spindle formation and function after nuclear envelope breakdown. Here, we used a proteomic approach to selectively identify proteins of this category and revealed 50 poorly characterised human gene products, among them the echinoderm microtubule-associated-protein-like gene product, EML3.