Motors involved in spindle assembly and chromosome segregation.

During the past two years, major advances have been made in our understanding of the role of motor proteins in chromosome-microtubule interactions in the spindle. The discovery of kinesin-like proteins (KLPs) associated with chromosome arms has shed some light on the mechanism of chromosome congression and the establishment of spindle bipolarity. Recent results also indicate that kinetochore KLPs may tether the ends of growing and shrinking microtubules to kinetochores during chromosome movements. Finally, new data indicate that phosphorylation of KLPs may be one of the mechanisms by which they are targeted to specific spindle domains.

The chaperonin CCT controls T cell receptor-driven 3D configuration of centrioles.

T lymphocyte activation requires the formation of immune synapses (IS) with antigen-presenting cells. The dynamics of membrane receptors, signaling scaffolds, microfilaments, and microtubules at the IS determine the potency of T cell activation and subsequent immune response. Here, we show that the cytosolic chaperonin CCT (chaperonin-containing TCP1) controls the changes in reciprocal orientation of the centrioles and polarization of the tubulin dynamics induced by T cell receptor in T lymphocytes forming an IS. CCT also controls the mitochondrial ultrastructure and the metabolic status of T cells, regulating the de novo synthesis of tubulin as well as posttranslational modifications (poly-glutamylation, acetylation, Δ1 and Δ2) of αß-tubulin heterodimers, fine-tuning tubulin dynamics. These changes ultimately determine the function and organization of the centrioles, as shown by three-dimensional reconstruction of resting and stimulated primary T cells using cryo-soft x-ray tomography. Through this mechanism, CCT governs T cell activation and polarity.

Chromosomes take the lead in spindle assembly.

Several kinesin-like motor proteins have recently been found associated with chromosome arms. They seem to be involved in the so-called 'polar ejection forces' that contribute to the congression of chromosomes on the metaphase plate, and at least one of them is essential for the maintenance of spindle bipolarity. The discovery of these molecules changes our view of the mechanism of spindle assembly and chromosome movement.

TPX2, A novel xenopus MAP involved in spindle pole organization.

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein-dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.

Localization of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a microtubule-associated protein, and dynein.

Xklp2 is a plus end-directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49-59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein-dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.

Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus.

The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

Heterotrimeric kinesin II is the microtubule motor protein responsible for pigment dispersion in Xenopus melanophores.

Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720-3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.

The mitotic spindle: a self-made machine.

The mitotic spindle is a highly dynamic molecular machine composed of tubulin, motors, and other molecules. It assembles around the chromosomes and distributes the duplicated genome to the daughter cells during mitosis. The biochemical and physical principles that govern the assembly of this machine are still unclear. However, accumulated discoveries indicate that chromosomes play a key role. Apparently, they generate a local cytoplasmic state that supports the nucleation and growth of microtubules. Then soluble and chromosome-associated molecular motors sort them into a bipolar array. The emerging picture is that spindle assembly is governed by a combination of modular principles and that their relative contribution may vary in different cell types and in various organisms.

A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity.

BACKGROUND: In eukaryotes, assembly of the mitotic spindle requires the interaction of chromosomes with microtubules. During this process, several motor proteins that move along microtubules promote formation of a bipolar microtubule array, but the precise mechanism is unclear. In order to examine the roles of different motor proteins in building a bipolar spindle, we have used a simplified system in which spindles assemble around beads coated with plasmid DNA and incubated in extracts from Xenopus eggs. Using this system, we can study spindle assembly in the absence of paired cues, such as centrosomes and kinetochores, whose microtubule-organizing properties might mask the action of motor proteins. RESULTS: We blocked the function of individual motor proteins in the Xenopus extracts using specific antibodies. Inhibition of Xenopus kinesin-like protein 1 (Xklp1) led either to the dissociation of chromatin beads from microtubule arrays, or to collapsed microtubule bundles on beads. Inhibition of Eg5 resulted in monopolar microtubule arrays emanating from chromatin beads. Addition of antibodies against dynein inhibited the focusing of microtubule ends into spindle poles in a dose-dependent manner. Inhibition of Xenopus carboxy-terminal kinesin 2 (XCTK2) affected both pole formation and spindle stability. Co-inhibition of XCTK2 and dynein dramatically increased the severity of spindle pole defects. Inhibition of Xklp2 caused only minor spindle pole defects. CONCLUSIONS: Multiple microtubule-based motor activities are required for the bipolar organization of microtubules around chromatin beads, and we propose a model for the roles of the individual motor proteins in this process.

Modeling the regulation of the bithorax complex in Drosophila melanogaster: the phenotypic effects of Ubx, abd-A and Abd-B heterozygotic larvae, and a homozygous Ubx- abd A hybrid gene.

As an intermediate step in the development of a defined quantitative model of pattern formation during Drosophila segmentation, we present here a model capable of predicting the experimentally determined levels of gene activity and their phenotypic consequences. In its present form, the model includes only four genes: the three genes of the bithorax complex (Ubx, abd-A and Abd-B) and Antennapedia. It is shown that the model is quite robust, predicting many properties in the behavior of these genes. A previously undescribed property is that all of these genes should phenotypically exhibit some kind of haploinsufficiency when present in only a single dose in the genetic background of the animal. This is shown both by the model and by a new method of quantitatively analyzing the differences in the more obvious cuticular features of the larvae, i.e., the patterns in the ventral denticle belts. The model is also capable of dealing with a complicated genetic situation, a hybrid gene of Ubx and abd-A produced by the C1 deletion.

Microgravity effects on the oogenesis and development of embryos of Drosophila melanogaster laid in the Spaceshuttle during the Biorack experiment (ESA).

The results obtained during the last successful flight of the Challenger Shuttle, in early November 1985, indicate that oogenesis and embryonic development of Drosophila melanogaster are altered in the absence of gravity. Two hundred forty females and ninety males, wild type Oregon R Drosophila melanogaster flies were flown in the Spaceshuttle during the 7-day D-1 mission and the embryos laid during the spaceflight were recovered and studied. Although some eggs developed into normal 1st instar larvae and many into late embryos in the 23 +/- 2 h collection periods throughout the flight, several interesting differences from the parallel ground and in-flight centrifuge controls were observed: 1) There was an increase in oocyte production and size. 2) There was a significant decrease in the number of larvae hatched from the embryonic cuticles in microgravity. 3) The majority of embryos were normally fertilized and at late stages of development, except in the space-flown containers in microgravity where a percentage of earlier stage embryos were recovered showing alterations in the deposition of yolk. 4) In correspondence with these results, at least 25% of the living embryos recovered from space failed to develop into adults. 5) Studies of the larval cuticles and those of the late embryos indicate the existence of alterations in the anterior, head and thoracic regions of the animals. 6) There was a delay in the development into adults of the embryos and larvae that had been subjected to microgravity and recovered from the space shuttle at the end of the flight. No significant accumulation of lethal mutations in any of the experimental conditions was detected as measured through the male to female ratio in the descendant generation. It seems that Drosophila melanogaster flies are able to sense and respond to the absence of gravity, changing several developmental processes even in very short space flights. The results suggest an interference with the distribution and/or deposition of the maternal components involved in the specification of the anterioposterior axis of the embryo.

Kinesin subfamily UNC104 contains a FHA domain: boundaries and physicochemical characterization.

By sequence analysis we show that the U104 domain found in the UNC104 subfamily of kinesins is a forkhead homology-associated domain (FHA). A combination of limited proteolysis, mass spectroscopy, and physicochemical analysis define this domain as a genuine autonomously folding domain. Our data show that the FHA domain is shorter than previously reported since the C-terminal alpha-helix is not part of its minimum core. Key amino acids postulated to recognize phosphorylated residues are conserved. These data suggest that the kinesin FHA domains are functional domains involved in protein-protein interactions regulated by phosphorylation.

Quantitative analysis of ventral denticular patterns of Drosophila melanogaster larvae and the regulation of the bithorax complex.

A quantitative model of the effect of the bithorax complex on segmentation is presented which could explain the known data of the spatiotemporal regulation of key gene complex during early Drosophila development, in relation to their effects on some of the segmentation landmarks. The model tries to put together the two different genetic levels, the genotypic and the phenotypic. At the genotypic level, a minimal cross-regulatory network of the different genes involved, Antp, Ubx, abd-A and Abd-B which explains the reported levels of expressions of these genes. At the phenotypic level, the pattern of the ventral denticle belts across the larva which are characteristics of the different segments have been compared by calculating a value of the degree of similarity in the case of the wild-type and several mutant combinations. Finally the two parts of the model are combined, showing that a satisfactory agreement between the two can be achieved. Therefore, this work is a first attempt to develop a method which will provide an explanatory solution of the old question in morphogenesis of how the phenotype is directed by the genotype of a cell or organism.

Insects as test systems for assessing the potential role of microgravity in biological development and evolution.

Gravity and radiation are undoubtedly the two major environmental factors altered in space. Gravity is a weak force, which creates a permanent potential field acting on the mass of biological systems and their cellular components, strongly reduced in space flights. Developmental systems, particularly at very early stages, provide the larger cellular compartments known, where the effects of alterations in the size of the gravity vector on living organisms can be more effectively tested. The insects, one of the more highly evolved classes of animals in which early development occurs in a syncytial embryo, are systems particularly well suited to test these effects and the specific developmental mechanisms affected. Furthermore, they share some basic features such as small size, short life cycles, relatively high radio-resistance, etc. and show a diversity of developmental strategies and tempos advantageous in experiments of this type in space. Drosophila melanogaster, the current biological paradigm to study development, with so much genetic and evolutionary background available, is clearly the reference organism for these studies. The current evidence on the effects of the physical parameters altered in space flights on insect development indicate a surprising correlation between effects seen on the fast developing and relatively small Drosophila embryo and the more slowly developing and large Carausius morosus system. In relation to the issue of the importance of developmental and environmental constraints in biological evolution, still the missing link in current evolutionary thinking, insects and space facilities for long-term experiments could provide useful experimental settings where to critically assess how development and evolution may be interconnected. Finally, it has to be pointed out that since there are experimental data indicating a possible synergism between microgravity and space radiation, possible effects of space radiation should be taken into account in the planning and evaluation of experiments designed to test the potential role of microgravity on biological developmental and evolution.

Chromosome motors on the move. From motion to spindle checkpoint activity.

Spindle assembly and chromosome segregation require the concerted activities of a variety of microtubule-dependent motors. This review focuses on our current knowledge of the roles played by the chromosome-associated motors during mitosis. While some appear to function conventionally in moving chromosomes along microtubules others seem to act in different ways. For example, by docking microtubules to chromosome arms, chromatin-associated motors prevent chromosome loss and participate in spindle formation and stability. Kinetochore motors participate in the formation of stable kinetochore fibers or in the control of microtubule dynamics and are involved in spindle checkpoint activity. Chromosome-associated motors thus appear to be key molecules that function in complementary ways to ensure the accuracy of chromosome segregation.

Multiple kinesin-like transcripts in Xenopus oocytes.

Recent evidence shows that kinesin-like proteins (Klps) form a very large multigene family. A recent study using the polymerase chain reaction (PCR) identified six new candidate Klps in Drosophila, making the total number of members of this family in Drosophila at least 11 (Stewart et al., 1991, Proc. Natl. Acad. Sci. USA 88, 4424-4427). The functional basis of this diversity is not clear. Different Klps could have cell type-specific functions, or they could perform different functions within the same cell type, or a mixture of both. To investigate the degree to which different Klps are expressed in the same cell, we chose the Xenopus oocyte. During oocyte differentiation, and in the egg, different types of microtubule-based motility occur; all are important to the normal development of the embryo after fertilization. Using PCR we identified and partially sequenced four novel Klp mRNAs from the Xenopus oocyte (denoted XKlps 1-4). Multialign sequence comparison suggests that one of them, XKlp3, may be the Xenopus counterpart of Drosophila Klp4. Similarly Xenopus Eg5 is closely related to Drosophila Klp2. Northern blot analysis reveals that the Xenopus XKlps have different patterns of expression during embryogenesis. These data show that at least four Klps can exist in the same cell and that they can be differentially regulated during early development, and suggest their differential function in oogenesis and early development.

Xklp2, a novel Xenopus centrosomal kinesin-like protein required for centrosome separation during mitosis.

We describe a novel Xenopus plus end-directed kinesin-like protein (KLP), Xklp2, localized on centrosomes throughout the cell cycle and on spindle pole microtubules during metaphase. Using mitotic spindles assembled in Xenopus egg extracts and different recombinant GST-Xklp2 mutants, we show that this motor is targeted to spindle poles through its C-terminal domain. Xklp2-truncated polypeptides lacking the motor domain block centrosome separation and disrupt preassembled metaphase spindles. Antibodies directed against the tail of Xklp2 have a similar effect. These results show that Xklp2 protein is required for centrosome separation and maintenance of spindle bipolarity. This study is an example of the application of the dominant negative mutant effect on spindle assembly in Xenopus egg extracts, demonstrating the usefulness of this approach in probing the function of proteins in this system.

[The possible role of cellular proto-oncogenes in early embryonic development]. / El posible papel de los protooncogenes celulares en el desarrollo embrionario temprano.

A brief review about the possible role of cell protooncogenes in embryonal development and neoplastic transformation is presented.