Interactive Fly, Drosophila

Three alpha-tubulin proteins contribute to microtubules during oogenesis and early embryogenesis in Drosophila melanogaster: alpha TUB84B, alpha TUB84D, and alpha TUB67C. alpha TUB67C is unique in two respects. It is a structurally divergent alpha-tubulin, sharing only 67% amino acid identity with the generic isotypes alpha TUB84B and alpha TUB84D, and its expression is exclusively maternal. The genetic analysis of the alpha Tub67C gene demonstrates that it is required for nuclear division in the oocyte and early embryo. Both meiosis and cleavage-stage mitoses are severely affected by mutations that result in a substantial decrease in the ratio of alpha TUB67C to alpha TUB84B plus alpha TUB84D. A large increase in this ratio, achieved by increasing the gene dosage of alpha Tub67C, has little or no effect on meiosis, but severely disrupts mitotic spindle function. Thus, both classes of alpha-tubulin isotype present in the mature oocyte, alpha TUB67C and alpha TUB84B/84D, are essential for normal spindle function in early Drosophila development. These alpha-tubulins provide the first example of tubulin isotypes known to be coexpressed and in which encoded variation is required for the normal function of a microtubule array (Matthews, 1993).

The ninefold microtubule symmetry of the eukaryotic basal body and motile axoneme has been long established. The basal body and axoneme contains a 9 + 2 arrangement of microtubules, with nine doublets surrounding two singlets. In Drosophila, these organelles contain distinct but similar ß-tubulin isoforms: basal bodies contain only ß1-tubulin, and only ß2-tubulin is used for assembly of sperm axonemes. A single alpha-tubulin functions throughout spermatogenesis. Thus, differences in organelle assembly reside in ß-tubulin. The ability of ß1 to function in axonemes has been tested and it was found that ß1 alone could not generate axonemes. Small sequence differences between the two isoforms therefore mediate large differences in assembly capacity, even though these two related organelles have a common evolutionarily ancient architecture. In males with equal ß1 and ß2, ß1 was co-incorporated at an equimolar ratio into functional sperm axonemes. When ß1 exceeds ß2, however, axonemes with 10 doublets are produced, an alteration unprecedented in natural phylogeny. Addition of the tenth doublet occurs by a novel mechanism, bypassing the basal body. It has been assumed that the instructions for axoneme morphogenesis reside primarily in the basal body, which normally serves as the axonemal template. These data reveal that ß-tubulin requirements for basal bodies and axonemes are distinct, and that key information for axoneme architecture resides in the axonemal ß-tubulin (Raff, 2000).

ß1 was expressed in post-mitotic cells under control of ß2 gene regulatory sequences. The transgenic construct drives ß1 expression to the same level as the endogenous ß2 gene, allowing for the examination of ß1 function with cellular tubulin pools at normal physiological levels. When ß1 was co-expressed with ß2 at gene doses such that ß1 comprises up to half of the ß-tubulin in the post-mitotic cells, spermatogenesis proceeds normally and ß1 is incorporated into sperm tail axonemes. Males with equimolar ß1 and ß2 in post-mitotic cells are fully fertile and sperm axonemes have wild-type morphology. Moreover, an equal gene dose of ß1 fully rescues defective microtubule function attributable to below-optimal tubulin pools, which cause diminished fecundity in males hemizygous for ß2 (Raff, 2000).

When ß1 is the only post-mitotic ß-tubulin, axoneme morphogenesis is profoundly defective; this shows that ß1 can not replace ß2. In ß1-only males, 9+0 axonemes are initiated at the basal body. Central pair microtubules, which in wild type invariably begin immediately distal to the basal body, were present in only 1 of 168 axonemes followed in serial sections in ß1-only males. The integrity of 9+0 axonemes is maintained for only a fraction of the normal 1.9mm sperm tail length (average, 20µm). Distally, axonemes either lose coherent organization or terminate. Disorganized axonemes in ß1-only males are remarkably like those assembled from ß2DeltaC, a truncated form of ß2 lacking the isotype-specific carboxyl terminus. Unlike ß2DeltaC, however, ß1 supports completion of accessory microtubules, and attachment of dynein arms and the spoke–linker complex. Thus, an acidic carboxyl terminus is necessary for these processes, but the ß2-specific carboxyl terminus is not required (Raff, 2000).

Differences in properties of ß1 and ß2 are striking when ß1 exceeds ß2 in the post-mitotic germ cells. Although equimolar amounts of ß1 and ß2 allow wild-type fertility, males are invariably sterile when ß1 is present with ß2 at a 2:1 ratio. Meiosis and cytoplasmic microtubules are normal in 2:1 males, but spermatids have a mixture of normal axonemes, axonemes lacking the central pair, and axonemes with 10 doublets -- a previously undescribed architecture. Initiation of 10-doublet axonemes was examined in serial sections. In all cases known, axonemal doublet microtubules are continuous with the basal body triplets. It was therefore hypothesized that when ß1 persists in the germ cells after its normal time of expression, basal bodies with 10 triplet microtubules are constructed, which would then template 10-doublet axonemes: this proved to be incorrect. Basal bodies observed in 2:1 males, just as in ß1-only males, have normal ninefold symmetry. Moreover, without exception, normal 9+2 axonemes are initiated at the basal body in 2:1 males. This 9+2 morphology is maintained for at least 40µm; 10-doublet axonemes are present only in more distal regions of the sperm tail. The tenth doublet is laterally inserted into axonemes de novo and appears abruptly, without obvious precursor structures. Loss of the tenth doublet and transition back to nine doublets was rarely observed. Only a few mature 10-doublet axonemes retained the central pair. A closed 10-doublet configuration appears to be incompatible with correct positioning of the central pair. Occasional axonemes containing more than 10 doublets are always in an opened configuration. The mechanism for association of the doublet microtubules with the spoke-linker apparatus in the internal axoneme 'cartwheel' structure can thus accommodate 10 doublets but not more (Raff, 2000).

Assembly at the basal body occurs with great fidelity. In 2:1 males, axoneme starts are 9+2. Similarly, in ß1-only males, axoneme starts have the canonical nine doublets. These observations suggest that the mechanism for initiating doublet microtubules from the basal body triplets constrains assembly -- either because of a direct 'templating' effect whereby the association of new tubulin dimers with pre-formed microtubules forces correct patterning even of imperfectly bonded subunits, or because of a high concentration near the basal body of other components involved in axoneme initiation. Defects in the central pair in 2:1 males, however, together with their absence in ß1-only males, suggests that assembly of the central pair apparatus and its association with the doublet microtubules is a key property of ß2 and other axonemal ß-tubulins, not possessed by ß1 (Raff, 2000).

The small sequence differences between ß1 and ß2 distinguish between two kinds of shared-wall microtubule assembly. ß1 has the capacity for basal body assembly, but these properties are not sufficient for axoneme morphogenesis. ß2 has the properties required for axoneme assembly, but it is inferred that ß2 cannot generate shared-wall microtubules de novo. Some of the instructions for each organelle are therefore intrinsic to the ß-tubulin subunits, and axoneme morphology is not solely dependent on the basal body template. It has been shown that ß-tubulin can specify microtubule protofilament substructure. Thus, to a hitherto unappreciated extent, information for a given microtubule-based structure can be built into the tubulins from which it is assembled (Raff, 2000).

This study demonstrates sorting of ß-tubulins during dimerization in the Drosophila male germ line. Different ß-tubulin isoforms exhibit distinct affinities for alpha-tubulin during dimerization. The data suggest that differences in dimerization properties are important in determining isoform-specific microtubule functions. The differential use of ß-tubulin during dimerization reveals structural parameters of the tubulin heterodimer not discernible in the resolved three-dimensional structure. The variable ß-tubulin carboxyl terminus, a surface feature in the heterodimer and in microtubules, and which is disordered in the crystallographic structure, is of key importance in forming a stable alpha-ß heterodimer. If the availability of alpha-tubulin is limiting, alpha-ß dimers preferentially incorporate intact ß-tubulins rather than a ß-tubulin missing the carboxyl terminus (ß2DeltaC). When alpha-tubulin is not limiting, ß2DeltaC forms stable alpha-ß heterodimers. Once dimers are formed, no further sorting occurs during microtubule assembly: alpha-ß2DeltaC dimers are incorporated into axonemes in proportion to their contribution to the total dimer pool. Co-incorporation of ß2DeltaC and wild-type ß2-tubulin results in nonmotile axonemes because of a disruption of the periodicity of nontubulin axonemal elements. These data show that the ß-tubulin carboxyl terminus has two distinct roles: (1) forming the alpha-ß heterodimer, important for all microtubules and (2) providing contacts for nontubulin components required for specific microtubule structures, such as axonemes (Hoyle, 2001).

The ß2-specific carboxyl terminus is essential for assembly of the sperm tail flagella, the only motile axoneme in Drosophila. The 15 carboxyl residues missing from ß2DeltaC include the axoneme motif present in all ß-tubulins incorporated into motile axonemes, as well as sites of posttranslational modification known to be important for axoneme motility. The axoneme motif specifies the central pair microtubules. Even when full-length ß2 makes up the majority of the ß-tubulin present, incorporation of stable ß2DeltaC into axoneme microtubules disrupts the organization of the central pair complex and the radial spokes. Studies of paralyzed flagella mutants of Chlamydomonas have shown these two axonemal structures to be involved in regulating axoneme motility. A nontubulin component found in the central pair complex of a wide range of species is the central pair projection. Transient contact between the radial spokes and the central pair projections is thought to play an important role in the regulation of dynein and the generation of the complex flagellar waveform (Hoyle, 2001).

The identity of the repeated element found in the Drosophila central pair complex is not known. However, its disruption by ß2DeltaC, as well as the disruption of the radial spokes, implies that both components are in contact with the carboxyl terminus of ß-tubulin in wild-type axonemes, either directly or indirectly through other protein-protein interactions. In these experiments, all nontubulin components present are wild type. When the radial spokes or central pair components are themselves mutant, as is the case with Chlamydomonas paralyzed flagellar mutants, the mutations are recessive. In contrast, when ß2 subunits lacking the carboxyl terminus are incorporated at random into axonemes, the consequence is a dominant disruption of axoneme motility. It appears that for each alpha84B-ß2DeltaC dimer incorporated into an axoneme microtubule, there is a stoichiometric loss of interaction with nontubulin components of the axoneme. When 30% of the ß-tubulin lacks the carboxyl terminus, the loss of organization becomes too great to permit axoneme function, and sperm become nonmotile. The higher the percentage of truncated ß-tubulin incorporated into the axoneme, the more severe are the defects. Axonemes from males in which ß2DeltaC is 50% of total ß-tubulin exhibit the same kind of defects that occurred in sterile males with 30% ß2DeltaC but to a much more marked degree, readily apparent in axoneme cross-sections (Hoyle, 2001).

The ß2-tubulin carboxyl terminus plays two distinct roles. In its first role, the acidic carboxyl terminus is important for producing a stable alpha-ß heterodimer. The generalized nature of this requirement is reflected by the fact that any acidic carboxyl terminus seems to work; ß1- and ß3-tubulin both form stable alpha-ß dimers in the male germ line. Thus, although the ß-tubulin carboxyl terminus is the hypervariable, isotype-defining region of the molecule, the wrong carboxyl terminus seems to be better than no carboxyl terminus. In a distinct second role, the carboxyl terminus is involved in interactions between intact microtubules and nontubulin components required to generate the architecture of specific microtubule-based structures. ß2DeltaC by itself fails to support any axonemal organization. However, not just any carboxyl terminus can support this function: other full-length Drosophila ß-tubulin isoforms cannot replace ß2 for axoneme function. In contrast, the carboxyl terminus is not required for generic microtubule functions: ß2DeltaC can support assembly of functional meiotic spindles as well as the cytoplasmic microtubules associated with elongation of the mitochondrial derivative. It should be noted that these generic functions can also be fully or partially supplied by other Drosophila isoforms (Hoyle, 2001).

These data demonstrate that competition between ß2DeltaC and intact ß-tubulins takes place during dimerization and not during subsequent microtubule assembly. However, competition between full-length ß-tubulin and ß2DeltaC could occur at several steps in the dimerization process. This work does not distinguish between competition for binding with cytosolic chaperonin, competition for cofactors of the dimer-making machine, or direct competition between ß2 and ß2DeltaC for alpha84B. However, the resolved three-dimensional structure of the alpha-ß dimer allows for the possibility that the unresolved ß-tubulin carboxyl residues are in contact with the alpha-tubulin moiety of the same alpha-ß dimer. Direct competition between ß-tubulins for binding to alpha is consistent with the finding that different ß-tubulin isoforms exhibit differential abilities to form alpha-ß dimers. This model suggests the possibility that alpha-tubulin residues contacted by the ß-tubulin carboxyl terminus play a direct role in stabilizing the alpha-ß dimer. The resolved structure predicts these alpha-tubulin residues to be non-carboxyl-terminal residues. As is the case with ß-tubulin, the alpha-tubulin carboxyl terminus is also unresolved in both the structures of the heterodimer and the microtubule. The 11 unresolved alpha residues are in position to contact ß-tubulin in another heterodimer, either in the same protofilament or perhaps in an adjacent protofilament. This leads to the prediction that the alpha-tubulin carboxyl terminus will be of relatively little importance in forming or stabilizing intrasubunit associations in the dimer but may be involved in interdimer associations in the protofilament substructure of microtubules (Hoyle, 2001).

Interacting proteins - Kinesins

The rapidly expanding kinesin family of microtubule motor proteins includes proteins that are involved in diverse microtubule-based functions in the cell. Phylogenetic analysis of the motor regions of the kinesin proteins reveals at least five clearly defined groups that are likely to identify kinesins with different roles in basic cellular processes. Two of the groups have overall sequence similarity, while two groups contain proteins that are related in overall structure or function but show no significant sequence similarity outside the motor domain. One of these groups consists only of kinesin proteins with predicted C-terminal motor domains; another includes only kinesins required for mitotic spindle bipolarity. Drosophila Nod, presently an ungrouped protein, may represent a class of kinesins that function as monomers, as do the myosin I proteins. The analysis indicates that many types of kinesin proteins exist in eukaryotic organisms. At least two of the five groups identified in this analysis are expected to be present in most, or all, eukaryotes (Goodson, 1994).

During Drosophila oogenesis (pre- to mid-vitellogenic stages) several morphogenetic determinants and other developmental factors synthesized in the nurse cells have been shown to accumulate in the oocyte. The transport processes are actin based some of the time, and at other times, microtubule based. By means of video-enhanced contrast time-lapse microscopy, the transport of cytoplasmic particles has been observed moving from the nurse cells through ring canals into the oocyte during oogenesis stages 6-10A. At stage 7, single particles have been observed moving into the previtellogenic oocyte. The particle transfer is strictly unidirectional and seemed to be selective, since only some individual particles move whereas other particles lying in the vicinity of the ring canals are not transported. The observed transport processes are inhibitable with 2,4-dinitrophenol, cytochalasin B or N-ethylmaleimide, but not with microtubule inhibitors, suggesting that transport is along the actin based cytoskeleton. At the beginning of vitellogenesis (stage 8), the selective translocation of particles through the ring canals becomes faster (up to 130 nm/second) and more frequent (about 1 particle/minute), whereas during mid-vitellogenesis (stages 9-10A) the velocity and the frequency of particle transport decreases again. Following their more or less rectilinear passage through the ring canals, the particles join a circular stream of cytoplasmic particles in the oocyte. This ooplasmic particle streaming starts at stage 6/7 at about 80 nm/second and some reversals of direction at the beginning. The particle stream in the oocyte is sensitive to colchicine and vinblastine, but not to cytochalasin B, presumably reflecting the rearrangement of ooplasmic microtubules. It has been proposed that during stages 7-10A, a selective transport of particles into the oocyte occurs through the ring canal along a polarized scaffold of cytoskeletal elements in which actin microfilaments are involved. This transport might be driven by a myosin-like motor molecule. Either attached to, or organized into, such larger particles or organelles, specific mRNAs and proteins might become selectively transported into the oocyte (Bohrmann, 1994).

A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH340) extends from the N terminus to amino acid 340. The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis in the absence of microtubules. The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules. The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules. One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules. These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule (Huang, 1994a).

The DKH392 construct includes an additional 52 amino acids beyond the minimal motor domain of 340 amino acid residues, previously characterized as DKH340. DKH340 is a monomer in solution, but DKH392 is a dimer. In the presence of adenosine 5-(beta,gamma-imido)triphosphate, DKH392 binds to microtubules with a stoichiometry of two head domains (one DKH392 dimer) per tubulin heterodimer, in contrast to the tight binding of one DKH340 per tubulin heterodimer. Electron microscopy indicates that both DKH340 monomers and DKH392 dimers decorate microtubules with a periodicity of 8 nm (Huang, 1994b).

Kid (kinesin-like DNA binding protein) is a novel member of the kinesin family of proteins. Nucleotide sequencing reveals that Kid is a 73 kDa protein and to some extent is related to the Drosophila nod gene product involved in chromosomal segregation during meiosis. A sequence similar to the microtubule binding motor domain of kinesin is present in the N-terminal half of the protein, also with the ability to bind to microtubules. Kid's C-terminal half contains a putative nuclear localization signal similar to that of Jun and is able to bind to DNA. Indirect immunofluorescence studies show that Kid colocalizes with mitotic chromosomes and that it is enriched in the kinetochore at anaphase. It has been proposed that Kid might play a role(s) in regulating the chromosomal movement along microtubules during mitosis (Tokai, 1996).

KLP3A (Kinesin-Like-Protein-at-3A) localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells (Williams, 1995).

The KLP61F gene product is essential for Drosophila development. Mutations in KLP61F display a mitotic arrest phenotype caused by a failure in the proper separation of duplicated centrosomes. Sequence analysis of KLP61F identified it as a member of the bimC family of kinesin-like microtubule motor proteins. KLP61F is distinct from KRP130, a kinesin-like protein recently purified from Drosophila embryos and suggested to be the product of the KLP61F gene. KLP61F possesses microtubule-stimulated ATPase and microtubule translocation activities in vitro. From early prophase through anaphase, KLP61F is coincident with the distribution of tubulin. Together these results confirm the existence of multiple bimC-like kinesins in Drosophila and suggest that KLP61F function is intrinsic to the mitotic spindle (Barton, 1995).

KLP68D, a new kinesin-like motor protein in the fly has a domain that shares significant sequence identity with the entire 340-amino acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled-coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different from those moved by kinesin heavy chain or other plus-end directed motors (Pasavento, 1994).

Pan-kinesin peptide antibodies were used to identify and isolate kinesin-related proteins (KRPs) from Drosophila melanogaster embryonic cytosol. These KRPs cosediment with microtubules (MTs) polymerized from cytosol treated with AMP-PNP (adenyl-5'-yl imidodiphosphate). One of them, KRP130, behaves as a homotetrameric complex composed of four 130-kDa polypeptide subunits that display a "slow" plus-end directed motor activity capable of moving single MTs at 0.04 microns/s. The 130-kDa subunit of KRP130 is related to Xenopus Eg5, a member of the BimC subfamily of kinesins. Therefore KRP130 appears to be the first Drosophila KRP (and the first member of the BimC subfamily in any organism) to be purified from native tissue as a multimeric motor complex (Cole, 1994).

A new member of the kinesin superfamily in Drosophila, KLP38B (kinesin-like protein at 38B) was isolated through its two-hybrid interaction with the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1). Recombinant KLP38B and PP1 associate in vitro. This is the first demonstration of direct binding of a kinesin-related protein to a regulatory enzyme. Though most closely related to the Unc-104 subfamily of kinesin-related proteins, KLP38B is expressed only in proliferating cells. KLP38B mutants show cell proliferation defects in many tissues. KLP38B is required for normal chromatin condensation, since embryos from KLP38B mutant mothers have undercondensed chromatin at metaphase and anaphase. This is the first time that a kinesin-related protein has been shown to have such a role. Incomplete lethality of a strong KLP38B allele suggests partial redundancy with one or more additional kinesin-related proteins (Alphey, 1997).

Members of the BimC subfamily of kinesin-related MT-motor proteins are believed to be essential for the formation and functioning of a normal bipolar spindle. KRP130 has an unusual ultrastructure. It consists of four kinesin-related polypeptides assembled into a bipolar aggregate with motor domains at opposite ends, analogous to a miniature myosin filament. Such a bipolar 'minifilament' could crosslink spindle MTs and slide them relative to one another. This is the first MT motor known to have a bipolar structure (Kashina, 1996).

Motor domains of the Drosophila minus-end-directed microtubule motor protein Non Claret Disjunction (NCD) saturate microtubule binding sites at a stoichiometry of approximately one motor domain per tubulin dimer. To determine the tubulin subunit(s) involved in binding to NCD, mixtures of NCD motor domain and MTs were treated with the cross-linker EDC. EDC treatment generates covalently cross-linked products of NCD and alpha-tubulin and of NCD and beta-tubulin, indicating that the NCD motor domain interacts with both alpha- and beta-tubulin. When the Drosophila kinesin motor domain protein is substituted for the NCD motor domain, cross-linked products of kinesin and alpha-tubulin and of kinesin and beta-tubulin are produced. EDC treatment of mixtures of NCD motor domain and unassembled tubulin dimers (or of kinesin motor domain and unassembled tubulin dimers) produce the same motor-tubulin products generated in the presence of MTs. These results indicate that kinesin family motors of opposite polarity interact with both tubulin monomers and support a model in which some portion of each protein's motor domain overlaps adjacent alpha- and beta-tubulin subunits (Walker, 1995).

Partial motor activity of NCD are sufficient for its mitotic, but not its meiotic, role. The partial loss of function mutant ncdD contains a single amino acid change in the putative microtubule binding region of the NCD motor domain. The maximum microtubule-stimulated ATPase activity of mutant protein is reduced three fold and a three fold greater concentration of microtubules is required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. Pelleting assays demonstrated that the binding of the mutant protein to microtubules is reduced in the absence of ATP, relative to wild-type (Moore, 1996).

The bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, it has been proposed that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs (Matthies, 1996).

The NOD kinesin-like protein is localized along the arms of meiotic chromosomes and is required to maintain the position of achiasmate chromosomes on the developing meiotic spindle. The localization of ectopically expressed NOD protein on mitotic chromosomes precisely parallels that observed for wild-type NOD protein on meiotic chromosomes. Moreover, the carboxyl-terminal half of the nod protein also binds to chromosomes when overexpressed in mitotic cells, whereas the overexpressed amino-terminal motor domain binds only to microtubules. Chromosome localization of the carboxyl-terminal domain of NOD depends upon an 82-amino acid region comprised of three copies of a sequence homologous to the DNA-binding domain of HMG 14/17 proteins. These data map the two primary functional domains of the NOD protein in vivo and provide a molecular explanation for the directing of the NOD protein to a specific subcellular component, the chromosome (Afshar, 1995).

Interacting proteins - The microtubule-associated protein Futsch

The localization of microtubules at the third instar synapse has been followed by visualizing tubulin (antitubulin immunoreactivity) or by following the localization of neuronally overexpressed tau-GFP. Futsch colocalizes with synaptic microtubules as identified by anti-alpha-tubulin. Colocalization is observed throughout the axon and synapse and always occurs at microtubule loops. Near perfect colocalization of Futsch and tubulin persists even when the microtubule organization is disrupted in hypomorphic futsch mutations, implying an association of Futsch with the microtubules. There is no immunoreactivity of Futsch with muscle microtubules, demonstrating that there is not antibody cross-reactivity in these double-staining experiments. Overexpressed tau-GFP also colocalizes with Futsch in synaptic cytoskeletal loop structures (Roos, 2000). These data are consistent with Futsch being associated with the microtubule cytoskeleton as suggested by in vitro data (Hummel, 2000). The supernatant from adult head extracts was incubated with polymerized microtubules at 37°C. Futsch is enriched in the subsequent pellet fraction in the presence of taxol-stabilized bovine microtubules. Thus, Futsch cofractionates with microtubules, and soluble Futsch can be precipitated with exogenously added vertebrate microtubules to adult fly head supernatants (Hummel, 2000).

Futsch associates with microtubules in vitro, and a genetic analysis demonstrates that Futsch is necessary for axonal and dendritic growth. Futsch colocalizes with microtubules and identifies cytoskeletal loops that traverse the lateral margin of select synaptic boutons. An apparent rearrangement of microtubule loop architecture occurs during bouton division, and a genetic analysis indicates that Futsch is necessary for this process. futsch mutations disrupt synaptic microtubule organization, reduce bouton number, and increase bouton size. These deficits can be partially rescued by neuronal overexpression of a futsch MAP1B homology domain. Genetic manipulations that increase nerve-terminal branching correlate with increased synaptic microtubule loop formation, and both processes require normal Futsch function. These synaptic microtubule loops are highly reminiscent of microtubule loops present within the growth cone, implying a fundamental similarity between the mechanisms of growth cone motility and the mechanisms of synaptic growth and branching (Roos, 2000).

Dyneins and Microtubule associated proteins (MAPS)

forward to beta Tubulin 56D Protein interactions part 2/2

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