Daniel J. Needleman

Evidence for an upper limit to mitotic spindle length.

Size specification of macromolecular assemblies in the cytoplasm is poorly understood [1]. In principle, assemblies could scale with cell size or use intrinsic mechanisms. For the mitotic spindle, scaling with cell size is expected, because the function of this assembly is to physically move sister chromatids into the center of nascent daughter cells. Eggs of Xenopus laevis are among the largest cells known that cleave completely during cell division. Cell length in this organism changes by two orders of magnitude ( approximately 1200 microm to approximately 12 microm) while it develops from a fertilized egg into a tadpole [2]. We wondered whether, and how, mitotic spindle length and morphology adapt to function at these different length scales. Here, we show that spindle length increases with cell length in small cells, but in very large cells spindle length approaches an upper limit of approximately 60 microm. Further evidence for an upper limit to spindle length comes from an embryonic extract system that recapitulates mitotic spindle assembly in a test tube. We conclude that early mitotic spindle length in Xenopus laevis is uncoupled from cell length, reaching an upper bound determined by mechanisms that are intrinsic to the spindle.

Nucleation and Transport Organize Microtubules in Metaphase Spindles

Spindles are arrays of microtubules that segregate chromosomes during cell division. It has been difficult to validate models of spindle assembly due to a lack of information on the organization of microtubules in these structures. Here we present a method, based on femtosecond laser ablation, capable of measuring the detailed architecture of spindles. We used this method to study the metaphase spindle in Xenopus laevis egg extracts and found that microtubules are shortest near poles and become progressively longer toward the center of the spindle. These data, in combination with mathematical modeling, imaging, and biochemical perturbations, are sufficient to reject previously proposed mechanisms of spindle assembly. Our results support a model of spindle assembly in which microtubule polymerization dynamics are not spatially regulated, and the proper organization of microtubules in the spindle is determined by nonuniform microtubule nucleation and the local sorting of microtubules by transport.

How does a millimeter-sized cell find its center?

Microtubules play a central role in centering the nucleus or mitotic in eukaryotic cells. However, despite common use of microtubules for centering, physical mechanisms can vary greatly, and depend on cell size and cell type. In the small fission yeast cells, the nucleus can be centered by pushing forces that are generated when growing microtubules hit the cell boundary. This mechanism may not be possible in larger cells, because the compressive force that microtubules can sustain are limited by buckling, so maximal force decreases with microtubule length. In a well-studied intermediate sized cell, the C. elegans fertilized egg, centrosomes are centered by cortex-attached motors that pull on microtubules. This mechanism is widely assumed to be general for larger cells. However, re-evaluation of classic experiments in a very large cell, the fertilized amphibian egg, argues against such generality. In these large eggs, movement of asters away from a part of the cell boundary that they are touching cannot be mediated by cortical pulling, because the astral microtubules are too short to reach the opposite cell boundary. A century ago, Herlant and Brachet discovered that multiple asters within a single egg center relative to the cell boundary, but also relative to each other. Here, we summarize current understanding of microtubule organization during the first cell cycle in a fertilized Xenopus egg, discuss how microtubule asters move towards the center of this very large cell, and how multiple asters shape and position themselves relative to each other.

Human microtubule-associated-protein tau regulates the number of protofilaments in microtubules: a synchrotron x-ray scattering study.

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from alphabeta heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius R(MT) of MTs with increasing Phi, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in R(MT) seems to be isoform independent. Significantly, R(MT) was observed to rapidly increase for 0 < Phi < 0.2 and saturate for Phi between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C(o)(MT) of MTs leading to changes in the curvature C(MT) (=1/R(MT)). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.

Cationic liposome–DNA complexes: from liquid crystal science to gene delivery applications

At present, there is an unprecedented level of interest in the properties and structures of complexes consisting of DNA mixed with oppositely charged cationic liposomes (CLs). The interest arises because the complexes mimic natural viruses as chemical carriers of DNA into cells in worldwide human gene therapy clinical trials. However, since our understanding of the mechanisms of action of CL–DNA complexes interacting with cells remains poor, significant additional insights and discoveries will be required before the development of efficient chemical carriers suitable for long-term therapeutic applications. Recent studies describe synchrotron X-ray diffraction, which has revealed the liquid crystalline nature of CL–DNA complexes, and three-dimensional laser-scanning confocal microscopy, which reveals CL–DNA pathways and interactions with cells. The importance of the liquid crystalline structures in biological function is revealed in the application of these modern techniques in combination with functional transfection efficiency measurements, which shows that the mechanism of gene release from complexes in the cell cytoplasm is dependent on their precise liquid crystalline nature and the physical and chemical parameters (for example, the membrane charge density) of the complexes. In §5, we describe some recent new results aimed at developing bionanotube vectors for gene delivery.

Spindle Fusion Requires Dynein-Mediated Sliding of Oppositely Oriented Microtubules

BACKGROUND Bipolar spindle assembly is critical for achieving accurate segregation of chromosomes. In the absence of centrosomes, meiotic spindles achieve bipolarity by a combination of chromosome-initiated microtubule nucleation and stabilization and motor-driven organization of microtubules. Once assembled, the spindle structure is maintained on a relatively long time scale despite the high turnover of the microtubules that comprise it. To study the underlying mechanisms responsible for spindle assembly and steady-state maintenance, we used microneedle manipulation of preassembled spindles in Xenopus egg extracts. RESULTS When two meiotic spindles were brought close enough together, they interacted, creating an interconnected microtubule structure with supernumerary poles. Without exception, the perturbed system eventually re-established bipolarity, forming a single spindle of normal shape and size. Bipolar spindle fusion was blocked when cytoplasmic dynein function was perturbed, suggesting a critical role for the motor in this process. The fusion of Eg5-inhibited monopoles also required dynein function but only occurred if the initial interpolar separation was less than twice the microtubule radius of a typical monopole. CONCLUSIONS Our experiments uniquely illustrate the architectural plasticity of the spindle and reveal a robust ability of the system to attain a bipolar morphology. We hypothesize that a major mechanism driving spindle fusion is dynein-mediated sliding of oppositely oriented microtubules, a novel function for the motor, and posit that this same mechanism might also be involved in normal spindle assembly and homeostasis.

Fast Microtubule Dynamics in Meiotic Spindles Measured by Single Molecule Imaging: Evidence That the Spindle Environment Does Not Stabilize Microtubules

Metaphase spindles are steady-state ensembles of microtubules. We used single molecule imaging to measure tubulin dynamics in spindles and non-spindle assemblies in Xenopus egg extract. Our results argue that the high density of microtubules in spindles is caused by local enhancement of nucleation, and not by local stabilization.

Active contraction of microtubule networks

Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction. DOI: http://dx.doi.org/10.7554/eLife.10837.001

Physical basis of spindle self-organization.

Significance The spindle segregates chromosomes during cell division and is composed of microtubules and hundreds of other proteins, but the manner in which these molecular constituents self-organize to form the spindle remains unclear. Here we use a holistic approach, based on quantitative measurements in spindles of the spatiotemporal correlation functions of microtubule density, orientation, and stresses, to identify the key processes responsible for spindle self-organization. We show that microtubule turnover and the collective effects of local microtubule interactions, mediated via motor proteins and cross-linkers, can quantitatively account for the dynamics and the structure of the spindle. We thus reveal the physical basis of spindle self-organization and provide a framework that may be useful for understanding cytoskeletal function in vivo. The cytoskeleton forms a variety of steady-state, subcellular structures that are maintained by continuous fluxes of molecules and energy. Understanding such self-organizing structures is not only crucial for cell biology but also poses a fundamental challenge for physics, since these systems are active materials that behave drastically differently from matter at or near equilibrium. Active liquid crystal theories have been developed to study the self-organization of cytoskeletal filaments in in vitro systems of purified components. However, it has been unclear how relevant these simplified approaches are for understanding biological structures, which can be composed of hundreds of distinct proteins. Here we show that a suitably constructed active liquid crystal theory produces remarkably accurate predictions of the behaviors of metaphase spindles—the cytoskeletal structure, composed largely of microtubules and associated proteins, that segregates chromosomes during cell division.

Pin-Hole Array Correlation Imaging: Highly Parallel Fluorescence Correlation Spectroscopy

In this work, we describe pin-hole array correlation imaging, a multipoint version of fluorescence correlation spectroscopy, based upon a stationary Nipkow disk and a high-speed electron multiplying charged coupled detector. We characterize the system and test its performance on a variety of samples, including 40 nm colloids, a fluorescent protein complex, a membrane dye, and a fluorescence fusion protein. Our results demonstrate that pin-hole array correlation imaging is capable of simultaneously performing tens or hundreds of fluorescence correlation spectroscopy-style measurements in cells, with sufficient sensitivity and temporal resolution to study the behaviors of membrane-bound and soluble molecules labeled with conventional chemical dyes or fluorescent proteins.