Self-organization Of Microtubules Research Articles

The use of dominant negative mutants to study the function of mitotic motors in the in vitro spindle assembly assay in Xenopus egg extracts.

The mitotic spindle is a transient cellular structure that distributes faithfully the chromosomes among the two daughter cells during mitosis. This highly dynamic bipolar structure assembles and disassembles periodically and its main structural components are microtubules (MTs) and chromosomes. Microtubule-dependent motors in particular members of the kinesin superfamily play an important role in directing the self-organization of microtubules into a bipolar spindle.

Methods for the study of centrosome-independent spindle assembly in Xenopus extracts

Publisher Summary This chapter discusses methods for the study of both centrosome-mediated and centrosome-independent spindle assembly in Xenopus egg extracts. The chapter describes simple assays for microtubule polymerization and organization, through the addition of dimethyl sulfoxide (DMSO) or purified centrosomes to form mitotic asters. The protocols described include microtubule aster reactions, bead spindle reactions, and inhibition of extract proteins. This system is ideal for studying mechanisms of self-organization, including microtubule nucleation around chromatin and motor-dependent function. The value of these methods extends beyond the study of microtubule self-organization, however. Both centrosome-mediated and centrosome-independent spindle assembly share mechanisms in common. Although centrosomes constitute visible and dominant organizing centers when they are present, motor functions are superimposed and are crucial for spindle organization, as they are when centrosomes are absent. Therefore, the study of both types of spindle assembly is important to understand the principles of microtubule organization in mitosis and meiosis.

Microtubule self-organization is gravity-dependent

Although weightlessness is known to affect living cells, the manner by which this occurs is unknown. Some reaction-diffusion processes have been theoretically predicted as being gravity-dependent. Microtubules, a major constituent of the cellular cytoskeleton, self-organize in vitro by way of reaction-diffusion processes. To investigate how self-organization depends on gravity, microtubules were assembled under low gravity conditions produced during space flight. Contrary to the samples formed on an in-flight 1 x g centrifuge, the samples prepared in microgravity showed almost no self-organization and were locally disordered.

A model for the self-organization of microtubules driven by molecular motors

We propose a two-dimensional model for the organization of stabilized microtubules driven by molecular motors in an unconfined geometry. In this model two kinds of dynamics are competing. The first one is purely diffusive, with an interaction between the rotational degrees of freedom, the second one is a local drive, dependent on microtubule polarity. As a result, there is a configuration dependent driving field. Applying a molecular field approximation, we are able to derive continuum equations. A study on the solutions shows nonequilibrium steady states. The presence and stability of such self-organized states are investigated in terms of entropy production. Numerical simulations confirm analytical results.

Chromophore-assisted light inactivation and self-organization of microtubules and motors.

Chromophore-assisted light inactivation (CALI) offers the only method capable of modulating specific protein activities in localized regions and at particular times. Here, we generalize CALI so that it can be applied to a wider range of tasks. Specifically, we show that CALI can work with a genetically inserted epitope tag; we investigate the effectiveness of alternative dyes, especially fluorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies of pulsed and continuous-wave illumination. We then use fluorescein-labeled hemagglutinin antibody fragments, together with relatively low-power continuous-wave illumination to examine the effectiveness of CALI targeted to kinesin. We show that CALI can destroy kinesin activity in at least two ways: it can either result in the apparent loss of motor activity, or it can cause irreversible attachment of the kinesin enzyme to its microtubule substrate. Finally, we apply this implementation of CALI to an in vitro system of motor proteins and microtubules that is capable of self-organized aster formation. In this system, CALI can effectively perturb local structure formation by blocking or reducing the degree of aster formation in chosen regions of the sample, without influencing structure formation elsewhere.

Early stages of spindle formation and independence of chromosome and microtubule cycles in Haemanthus endosperm.

We analyzed transformation of the interphase microtubular cytoskeleton into the prophase spindle and followed the pattern of spindle axis determination. Microtubules in endosperm of the higher plant Haemanthus (Scadoxus) were stained by the immunogold and immunogold silver-enhanced methods. Basic structural units involved in spindle morphogenesis were "microtubule converging centers." We emphasized the importance of relative independence of chromosomal and microtubular cycles, and the influence of these cycles on the progress of mitosis. Cells with moderately desynchronized cycles were functional, but extreme desynchronization led to aberrant mitosis. There were three distinct phases of spindle development. The first one comprised interphase and early to mid-prophase. During this phase, the interphase microtubule meshwork radiating from the nuclear surface into the cytoplasm rearranged and formed a dense microtubule cage around the nucleus. The second phase comprised mid to late prophase, and resulted in the formation of normal (bipolar) or transitory aberrant (apolar or multipolar) prophase spindles. The third phase comprised late prophase with prometaphase. The onset of prometaphase was accompanied by a rapid association of microtubule converging centers with kinetochores. In this stage aberrant spindles transformed invariably into bipolar ones. Lateral association of a few bipolar kinetochore fibers at early prometaphase established the core of the bipolar spindle and its alignment. We concluded that (1) spindle formation is a largely independent microtubular process modified by the chromosomal/kinetochore cycle; and (2) the initial polarity of the spindle is established by microtubule converging centers, which are a functional substitute of the centrosome/MTOC. We believe that the dynamics of microtubule converging centers is an expression of microtubule self-organization driven by motor proteins as proposed by Mitchison [1992: Philos. Trans. R. Soc. Lond. B. 336:99].

Self-organization of microtubules and motors.

Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly 'pathways.

Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization.

In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end-directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts.

Functional nuclei and mitotic spindles are shown to assemble around DNA-coated beads incubated in Xenopus egg extracts. Bipolar spindles assemble in the absence of centrosomes and kinetochores, indicating that bipolarity is an intrinsic property of microtubules assembling around chromatin in a mitotic cytoplasm. Microtubules nucleated at dispersed sites with random polarity rearrange into two arrays of uniform polarity. Spindle-pole formation requires cytoplasmic dynein-dependent translocation of microtubules across one another. It is proposed that spindles form in the absence of centrosomes by motor-dependent sorting of microtubules according to their polarity.