betaTubulin56D (ß1 tubulin)
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).
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).
Hypoparathyroidism, mental retardation and facial dysmorphism (HRD) is a fatal developmental disease caused by mutations in tubulin-specific chaperone E (TBCE). A mouse Tbce mutation causes progressive motor neuronopathy. To dissect the functions of TBCE and the pathogenesis of HRD, mutations were generated in Drosophila tbce, and its expression was manipulated in a tissue-specific manner. Drosophila tbce nulls are embryonic lethal. Tissue-specific knockdown and overexpression of tbce in neuromusculature resulted in disrupted and increased microtubules, respectively. Alterations in TBCE expression also affected neuromuscular synapses. Genetic analyses revealed an antagonistic interaction between TBCE and the microtubule-severing protein Spastin. Moreover, treatment of muscles with the microtubule-depolymerizing drug nocodazole implicated TBCE as a tubulin polymerizing protein. Taken together, these results demonstrate that TBCE is required for the normal development and function of neuromuscular synapses and that it promotes microtubule formation. As defective microtubules are implicated in many neurological and developmental diseases, this work on TBCE may offer novel insights into their basis (Jin, 2009).
Microtubules (MTs), one of the major building blocks of cells, play a crucial role in a diverse array of biological functions including cell division, cell growth and motility, intracellular transport and the maintenance of cell shape. As MTs are important in all eukaryotes, it is not surprising that defects in MTs are associated with a number of severe human diseases, including Fragile X mental retardation and autosomal dominant hereditary spastic paraplegia (AD-HSP). MTs are formed by polymerization of tubulin heterodimers consisting of one α- and one β-tubulin polypeptide. The formation of α-β tubulin heterodimers is mediated by a group of five tubulin chaperones, TBCA-TBCE (see Domain analysis of the tubulin cofactor system: a model for tubulin folding and dimerization). TBCA and TBCD assist in the folding of β-tubulin, whereas TBCB and TBCE facilitate the folding of α-tubulin (Jin, 2009 and references therein),
A group of rare, recessive and fatal congenital diseases, collectively called hypoparathyroidism, mental retardation and facial dysmorphism (HRD), is caused by mutations in the gene encoding TBCE (Parvari, 2002). TBCE contains three functional domains: a glycine-rich cytoskeleton-associated protein domain (CAP-Gly) that binds α-tubulin, a series of leucine-rich repeats (LRR), and an ubiquitin-like (UBL) domain; the latter two mediate protein-protein interactions. Identification of the HRD disease gene revealed a 12 bp deletion in TBCE that leads to the expression of a mutated TBCE protein lacking four amino acids in the CAP-Gly domain. The mutation causes lower MT density at the MT organizing center, perturbed MT polarity and decreased precipitable MT, while total tubulin remains unchanged. Remarkably, overexpression of TBCE in cultured cells also results in disrupted MTs. Thus, both loss-of-function mutations and overexpression of TBCE disrupt the MT network in mammalian systems (Jin, 2009 and references therein).
Two independent studies have demonstrated that a Trp524Gly substitution at the last residue of mouse TBCE results in progressive motor neuronopathy (PMN), which has been widely used as a model for human motor neuron diseases. Similar to what has been reported for cells from human HRD patients, the point mutation in mouse Tbce leads to a reduced number of MTs in axons. Isolated motor neurons from mutant mice exhibit shorter axons and irregular axonal swellings. More specifically, axonal MTs are lost progressively from distal to proximal, which correlates with dying-back axonal degeneration in mutant mice. This demonstrates a mechanistic link between TBCE-mediated tubulin polymerization and neurodegeneration (Jin, 2009 and references therein).
TBCE is well conserved across species, from yeast to human. Genetic analyses of the TBCE homolog in S. pombe, Sto1P, show that it is essential for viability and that it plays a crucial role in the formation of cytoplasmic MTs and in the assembly of mitotic spindles. S. cerevisiae mutants of the TBCE homolog PAC2 show increased sensitivity to the MT-depolymerizing agent benomyl. Similarly, tbce mutants of Arabidopsis have defective MTs, leading to embryonic lethality (Jin, 2009 and references therein).
The Drosophila genome contains a TBCE ortholog, listed as CG7861 in FlyBase, but no studies of it have been reported. To gain a mechanistic insight into the in vivo functions of TBCE, different mutations were introduced into Drosophila tbce. Drosophila tbce nulls are embryonic lethal, indicating that it is an essential gene. The developmental, physiological and pharmacological consequences with regard to neuromuscular synapses and MT formation were examined when the expression of TBCE was altered specifically in neurons or muscles using the UAS-Gal4 system. It was found that TBCE is required for the normal development and function of neuromuscular synapses and that it promotes MT formation in vivo (Jin, 2009).
How MTs functions at synapses is poorly understood. Futsch, a MT-associated protein, stabilizes MTs in presynaptic neurons, and futsch mutants show reduced bouton number and increased bouton size, whereas spastin mutants have the opposite phenotype of increased bouton number but decreased bouton size. This analyses show that TBCE plays a role at synapses. Except for the presynaptic overexpression of tbce, all other manipulations of tbce at either side of the NMJ synapse caused increased branching number, increased bouton number and decreased bouton size, demonstrating that tbce is required for normal NMJ synapse development. Given the dramatic MT alterations on both pre- and postsynaptic sides, the NMJ phenotypes appear subtle. The seemingly conflicting result that both overexpression and knockdown of tbce on the postsynaptic side led to similar phenotypes in synapse development supports an existing hypothesis that abnormal synaptic growth results from the disruption of MT dynamics, rather than from an alteration in the absolute quantity of MTs (Jin, 2009).
Increased neurotransmission, reflected in both EJP and mEJP amplitude, was observed upon presynaptic alteration of tbce expression, whereas postsynaptic manipulations of tbce showed normal neurotransmission. This suggests that synaptic neurotransmission is sensitive to pre-but not postsynaptic MT alteration, although postsynaptic alterations of tbce had a significant effect on synapse development. Interestingly, both overexpression and knockdown of tbce on the presynaptic side led to a similar increase in both EJP and mEJP amplitude. The increased EJP amplitude observed upon presynaptic alterations of TBCE might be accounted for by increased mEJP amplitude. The increase in mEJP amplitude could be caused by an increase in presynaptic vesicle size, an increase in the concentration of vesicular glutamate, or an increase in postsynaptic glutamate receptor sensitivity. It is interesting to note that the mEJP is also increased in both Fmr1-null and Fmr1-overexpression NMJ synapses. However, the exact mechanism by which TBCE, and other MT regulators, affect neurotransmission remains to be elucidated (Jin, 2009).
Genetic analyses revealed an antagonistic interaction between TBCE and Spastin. TBCE promotes MT formation, whereas Spastin severs MTs. Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a heterogeneous group of neurodegenerative disorders characterized by progressive and bilateral spasticity of the lower limbs, with specific degeneration of the longest axons in the CNS. Forty to fifty percent of all AD-HSP cases are caused by mutations in spastin. However, the MT-related pathology of human patients with spastin mutation has not been documented (Jin, 2009).
Overexpression of spastin in Drosophila neuromusculature and in cultured cells causes dramatically fragmented and reduced MTs. Surprisingly, morphologically normal muscles are present in patients with spastin mutations, although large-scale disruption of MT pathways was detected at the molecular level. No MT defects were reported in a mouse model in which the endogenous spastin is truncated. Similarly, spastin-null mutants of Drosophila show no dramatic change in MT appearance in muscles, suggesting that Spastin plays a fine-tuning role in MT dynamics. Indeed, spastin nulls are late pupal lethal with a few adult escapers, further confirming a subtle role for Spastin in MT regulation. By comparison, tbce nulls are embryonic lethal, whereas knockdown of tbce leads to a dramatically reduced MT network in Drosophila neuromusculature. Thus, in contrast to the nuanced role of endogenous Spastin, TBCE plays a crucial role in MT formation (Jin, 2009).
Although Drosophila possesses a TBCE ortholog, no previous studies of it have been reported. This work shows that tbce is essential for early neuromuscular development in Drosophila. In vivo evidence is provided demonstrating that Drosophila TBCE is both required and sufficient for MT formation, supporting early in vitro biochemical studies that showed that TBCE assists in α-β-tubulin heterodimer formation (Jin, 2009 and references therein).
Overexpression of tbce produced increased MTs. This is the first report of increased MT formation when a tubulin chaperone is overexpressed, and is contrary to reports in other systems. Overexpression of human TBCE in cultured cells leads to complete disruption of MTs, as does overexpression of a TBCE-like protein. It was further hypothesized that the UBL domains present in TBCE and the TBCE-like protein might contribute to the degradation of tubulin via the proteasomal pathway. In addition, the overexpression of other tubulin chaperones, such as TBCD, results in a similar disruption of MTs. These in vivo data are consistent with the early in vitro observation that TBCD or TBCE in excess destroys tubulin heterodimers by sequestering the bound tubulin subunit, leading to the destabilization of the freed partner subunit. It is thus believed that in addition to assisting in the folding pathway, TBCE also interacts with native tubulins to disrupt α-β-tubulin heterodimers. The discrepancy between the overexpression result and findings of others could have several explanations: (1) the use of different experimental systems: transgenic animals in this work and cultured cells in other studies; (2) different systems might have different expression levels of tbce, leading to varying effects on MTs; (3) Drosophila and human TBCE might have diverged functions. Further analyses are needed to reconcile the conflicts in the effects of TBCE overexpression in these different systems. In general, however, tbce mutant phenotypes are consistent in all species examined so far, from yeast to human, indicating that the function of TBCE in promoting MT formation has been well-conserved throughout evolution (Jin, 2009).
Microtubules (MTs) are essential for cell division, shape, intracellular transport, and polarity. MT stability is regulated by many factors, including MT-associated proteins and proteins controlling the amount of free tubulin heterodimers available for polymerization. Tubulin-binding cofactors are potential key regulators of free tubulin concentration, since they are required for alpha-beta-tubulin dimerization in vitro. This paper shows that mutation of the Drosophila tubulin-binding cofactor B (TBCB) affects the levels of both alpha- and beta-tubulins and dramatically destabilizes the MT network in different fly tissues. However, this study found that dTBCB is dispensable for the early MT-dependent steps of oogenesis, including cell division, and that dTBCB is not required for mitosis in several tissues. In striking contrast, the absence of dTBCB during later stages of oogenesis causes major defects in cell polarity. dTBCB is required for the polarized localization of the axis-determining mRNAs within the oocyte and for the apico-basal polarity of the surrounding follicle cells. These results establish a developmental function for the dTBCB gene that is essential for viability and MT-dependent cell polarity, but not cell division (Baffet, 2012).
The TBCB protein is part of an evolutionarily conserved tubulin-folding pathway crucial for the formation of the tubulin heterodimers. Indeed, TBCs are thought to regulate MT dynamics by modulating the concentration of dimers available for polymerization (Lopez-Fanarraga, 2001). Using Drosophila as a model system, this study looked at the requirement for TBCB during development. dTBCB wsa shown to be required for MT integrity and is essential for viability, MT-associated transport, and cell polarity (Baffet, 2012).
TBCs are mostly cytoplasmic proteins, in accordance with their tubulin-chaperoning function. However, TBCE also accumulates at the Golgi apparatus of motor neurons, where it is essential for axonal tubulin routing (Schaefer, 2007). TBCD is concentrated at the centrosome, midbody, and cell junctions, where it participates in centriologenesis, spindle organization, cell abscission, and epithelial cell structure (Cunningham, 2008; Shultz, 2008; Fanarraga, 2010). The current results indicate that dTBCB is largely cytoplasmic but also partly overlaps with MTs, corroborating some previous studies in different species. This partial colocalization is particularly clear in developing embryos and in S2 cells. Such distribution is not observed with dTBCE, the α-TBC interacting with TBCB. This possibly indicates that dTBCB fulfills additional functions independent of the other TBCs. Further experiments willbe required to address these potential functions (Baffet, 2012).
dTBCB is required for the integrity of the MT network in different tissues. This is consistent with data obtained in yeast knockout, but not in mammalian knockdown cells. TBCB has been shown, together with TBCE, to stimulate tubulin heterodimer dissociation in vitro. In accordance with this, it was found that strong dTBCB overexpression in S2 cells triggers MT depletion. The increased MT density observed in mammalian microglia, after TBCB depletion by RNAi, might therefore be due to a reduced tubulin heterodimer dissociation. The different phenotypes reported in previous studies might be due to species specificity or to the efficiency of the technical approaches, with the mutational approach probably being more efficient than RNAi. It is proposed that severely impaired tubulin dimerization is the cause of the strong MT defects in yeast and Drosophila. In mammalian knockdown cells, however, a sufficient pool of tubulin heterodimers is produced, and only the dissociation of the tubulin heterodimer seems affected. Altogether these results suggest that TBCB is required for tubulin dimerization and for tubulin heterodimer dissociation in vivo. The latter function might be more sensitive to variations in TBCB concentration and could be used to fine-tune MT polymerization in a spatiotemporal manner (Baffet, 2012).
The α-TBC TBCE, a direct partner of TBCB, is required for viability and MT formation in Drosophila but does not affect α-tubulin levels (Jin, 2009). In dTBCB1 mutant egg chambers and larvae, however, a strong reduction was observed of α-tubulin levels. This suggests that the monomeric α-tubulins, which failed to bind dTBCB and to dimerize, are unstable. This is consistent with the fact that monomeric tubulins have been observed to be very unstable molecules in vitro. It was also observed that the β-tubulin levels were reduced in dTBCB1, although TBCB is known to associate specifically with α-tubulin. This suggests that the monomeric form of β-tubulin in Drosophila is unstable, even in the presence of its putative dedicated chaperone, dTBCA. It is interesting to note that knocking down mammalian TBCA also induces a decrease in both α- and β-tubulin levels. In dTBCB1 mutant tissues, the observed tubulin levels decrease most likely causes the MT destabilization. However, dTBCB does not seem absolutely required to form tubulin heterodimers in vivo, since wa significant pool of dimers is still present in mutant extracts. Overall the current results suggest that dTBCB enhances tubulin dimer formation in vivo to promote MT assembly (Baffet, 2012).
In plants and yeasts, TBCB is essential for cell division. In flies, dTBCB is expressed throughout cell cycle, and during mitosis it colocalizes to some extend with the MT spindle.Surprisingly, dTBCB knockout in diverse tissues does not prevent cell division, suggesting that dTBCB is not essential for mitosis. It was observed that mitotic spindles do form in mutant neuroblasts and allow mitosis, even if the overall cell cycle is slower than normal. Therefore the remaining levels of α- and β-tubulin are sufficient to fulfill cell division. Similarly, depletion of Drosophila TBCE decreases MT levels without precluding cell proliferation proceeding in embryos (Jin, 2009). Likewise, even though a null allele of α-tubulin 84B is cell-lethal, a hypomorphic mutation can affect larval viability without preventing cell proliferation). This suggests that mitosis in flies is a robust process, resistant to a significant decrease in α- and β-tubulin amounts. It is possible that, at least in Drosophila, TBCs are not essentials to sustain the MT polymerization level necessary for mitosis (Baffet, 2012).
This study has found that dTBCB is required for cell polarity in the ovary. In the oocyte and in the overlying epithelial follicle cells, the MT-based transport is particularly important for the asymmetric distribution of determinants. The drop in MT level observed in dTBCB mutant cells correlates with defects in localization of apico-lateral polarity proteins in epithelial cells, defects of mRNAs, and nucleus-polarized transport in oocyte at midoogenesis. However, low MT density does not trigger obvious polarity defects in early oogenesis polarization steps, since Orb accumulation occurs normally in mutant oocytes. It therefore seems that there is an interesting differential requirement for dTBCB between early and late stages of oogenesis that is not due to the persistence of the protein. This may point to a higher robustness of the MT-dependent processes in early stages of oogenesis: a partial depolymerization of the MT network may affect late but not early events. Early polarity maintenance could be due to the smaller size of the early cysts that probably need a lower MT-nucleation activity to produce a functional MT network (Baffet, 2012).
The simultaneous decreases of the expression levels of dTBCB and the ubiquitously expressed α-tubulin 84B prevent egg hatching and cause cell polarity defects in ovarian epithelial cells, suggesting that dTBCB regulates polarity most likely by controlling tubulin/MT integrity. Interestingly, this heteroallelic combination does not impair the formation/division of the follicle cell monolayer but affects its apico-basal polarity. These results confirm that mitotic cells are less sensitive to MT destabilization than interphasic cells, probably because of the much higher MT nucleation activity during mitosis. In accordance with this, it is interesting to note that nocodazol treatment depolymerizes inter phasic MT networks much more efficiently than mitotic spindle (Baffet, 2012).
In humans, the presence of a mutated α-tubulin defective for TBCB binding has been correlated with the brain malformation lissencephaly, while high dTBCB levels have been reported in breast tumors . The results of this study may be of significant importance in understanding the molecular basis of the development of these pathologies (Baffet, 2012).
Prefoldin is a molecular chaperone complex that regulates tubulin function in mitosis. This study shows that Prefoldin depletion results in disruption of neuroblast polarity, leading to neuroblast overgrowth in Drosophila larval brains. Interestingly, co-depletion of Prefoldin and Partner of Inscuteable (Pins) leads to the formation of gigantic brains with severe neuroblast overgrowth, despite that Pins depletion alone results in smaller brains with partially disrupted neuroblast polarity. This study shows that Prefoldin acts synergistically with Pins to regulate asymmetric division of both neuroblasts and Intermediate Neural Progenitors (INPs). Surprisingly, co-depletion of Prefoldin and Pins also induces dedifferentiation of INPs back into neuroblasts, while depletion either Prefoldin or Pins alone is insufficient to do so. Furthermore, knocking down either α-tubulin or β-tubulin in pins- mutant background results in INP dedifferentiation back into neuroblasts, leading to the formation of ectopic neuroblasts. Overexpression of α-tubulin suppresses neuroblast overgrowth observed in prefoldin pins double mutant brains. These data elucidate an unexpected function of Prefoldin and Pins in synergistically suppressing dedifferentiation of INPs back into neural stem cells (Zhang, 2016).
Control of tissue homeostasis is a central issue during development. The neural stem cells, or neuroblasts, of the Drosophila larval brain is an excellent model for studying stem cell homeostasis. Asymmetric division of neuroblasts generates a self-renewing neuroblast and a different daughter cell that undergoes differentiation pathway to produce neurons or glia. Following each asymmetric division, apical proteins such as aPKC are segregated into the neuroblast daughter and function as 'proliferation factor', while basal proteins are segregated into a smaller daughter cell to act as 'differentiation factors'. At the onset of mitosis, the Partitioning defective (Par) protein complex that is composed of Bazooka (Baz)/Par3, Par6 and atypical protein kinase C (aPKC) is asymmetrically localized at the apical cortex of the neuroblast. Other apical proteins including Partner of Inscuteable (Pins), the heterotrimeric G protein Gαi, and Mushroom body defect (Mud) also accumulate at the apical cortex through an interaction of Inscuteable (Insc) with Par protein complex. Apical proteins control basal localization of cell fate determinants Numb, Prospero (Pros), Brain tumor (Brat) and their adaptor proteins Miranda (Mira) and Partner of Numb (Pon) that are segregated into the ganglion mother cell (GMC) following divisions. Apical proteins and their regulators also control mitotic spindle orientation to ensure correct asymmetric protein segregation at telophase. Several centrosomal proteins, Aurora A, Polo and Centrosomin, regulate mitotic spindle orientation (Zhang, 2016).
There are at least two different types of neuroblasts that undergo asymmetric division in the larval central brain. Perturbation of asymmetric division in either type of neuroblast can trigger neuroblast overproliferation and/or the induction of brain tumors. The majority of neuroblasts are type I neuroblasts that generate a neuroblast and a GMC in each division, while type II neuroblasts generate a neuroblast and an intermediate neural progenitor (INP), which undergoes three to five rounds of asymmetric division to produce GMCs. Ets transcription factor Pointed (PntP1 isoform), exclusively expressed in type II neuroblast lineages, promotes the formation of INPs. Failure to restrict the self-renewal potential of INPs can lead to dedifferentiation, allowing INPs to revert back into 'ectopic neuroblasts'. Notch antagonist Numb and Brat function cooperatively to promote the INP fate. Loss of brat or numb leads to 'ectopic type II neuroblasts' originating from uncommitted immature INPs that failed to undergo maturation. A zinc-finger transcription factor Earmuff functions after Brat and Numb in immature INPs to prevent their dedifferentiation. Earmuff also associates with Brahma and HDAC3, which are involved in chromatin remodeling, to prevent INP dedifferentiation. However, the underlying mechanism by which INPs possess limited developmental potential is largely unknown (Zhang, 2016).
Prefoldin (Pfdn) was first identified as a hetero-hexameric chaperone consisting of two α-like (PFDN3 and 5) and four β-like (PFDN 1, 2, 4 and 6) subunits, based on its ability to capture unfolded actin (Vainberg, 1998). Prefoldin promotes folding of proteins such as tubulin and actin by binding specifically to cytosolic chaperonin containing TCP-1 (CCT) and by directing target proteins to it. The yeast homologs of Prefoldin 2–6, named GIM1-5 (genes involved in microtubule biogenesis) are present in a complex that facilitates proper folding of α-tubulin and γ-tubulin. All Prefoldin subunits are phylogenetically conserved from Archaea to Eukarya. Structural study of the Prefoldin hexamer from the archaeum M. thermoautotrophicum showed that Prefoldin forms a jellyfish-like shape consisting of a double β barrel assembly with six long tentacle-like coiled coils that participate in substrate binding. The function of Prefoldin as a chaperone has also been illustrated in lower eukaryotes like C. elegans, in which loss of prefoldin resulted in defects in cell division due to reduced microtubule growth rate. Depletion of PFDN1 in mice displayed cytoskeleton-related defects, including neuronal loss and lymphocyte development defects. The only Prefoldin subunit in Drosophila that has been characterized to date, Merry-go-round (Mgr), the Pfdn3 subunit, cooperates with the tumor suppressor Von Hippel Lindau (VHL) to regulate tubulin stability (Delgehyr, 2012). However, the functions of Prefoldin in the nervous system remain elusive (Zhang, 2016).
This study describes the critical role of evolutionarily-conserved Prefoldin complex in regulating neuroblast and INP asymmetric division and suppressing INP dedifferentiation. Mutants for two Prefoldin subunits, Mgr and Pfdn2, displayed neuroblast overgrowth with defects in cortical polarity of Par proteins and microtubule-related abnormalities. Interestingly, co-depletion of Pins in mgr or pfdn2 mutants led to massive neuroblast overgrowth. Prefoldin and Pins synergistically regulate asymmetric division of both neuroblasts and INPs. Surprisingly, they also synergistically suppress dedifferentiation of INPs back into neuroblasts. Knocking down tubulins in pins mutant background resulted in severe neuroblasts overgrowth, mimicking that caused by co-depletion of Prefoldin and Pins. These data provide a new mechanism by which Prefoldin and Pins regulates neural stem cell homeostasis through regulating tubulin stability in both neuroblasts and INPs (Zhang, 2016).
pfdn2/CG6302, encoding a Prefoldin β-like subunit, was identified from a RNA interference (RNAi) screen in larval brains. Ectopic neuroblasts labeled by a neuroblast marker, Deadpan (Dpn), were formed upon knocking down pfdn2 under a neuroblast driver insc-Gal4. Only one neuroblast was observed in control type I neuroblast lineages using insc-Gal4 and type II neuroblast lineages using worniu-Gal4 with asense (ase)-Gal80. In contrast, upon pfdn2 RNAi excess neuroblasts were observed in both type I neuroblast lineages and type II neuroblast lineages, respectively. To verify the function of Pfdn2 in neuroblasts, a putative hypomorphic allele of pfdn2, pfdn201239, was analyzed that has a P element inserted at the 5′ untranslated region (UTR) of pfdn2. Hemizygous larval brains of pfdn201239 over Df(3L)BSC457 (referred to as pfdn2− thereafter) displayed 235.3 ± 31.7 neuroblasts per brain hemisphere, suggesting that Pfdn2 inhibits the formation of ectopic neuroblasts in larval brains. Consistently, an increase of EdU (5-ethynyl-2′-deoxyuridine)-incorporation was also observed in pfdn2− mutants compared to the control. To generate pfdn2 null alleles, a P element, EY06124, was mobilized. Its imprecise excision yielded two loss-of-function alleles, pfdn2Δ10 and pfdn2Δ17, both deleting the entire opening reading frame (ORF) of pfdn2. pfdn2Δ10 and pfdn2Δ17 mutants survive to pupal stage and display strong phenotypes with ectopic neuroblasts labeled by Dpn. These phenotypes in pfdn2Δ10 and pfdn2Δ17 mutant brains can be fully rescued by overexpression of wild-type pfdn2 or pfdn2-Venus transgene. Pfdn2 is abundantly expressed in neuroblasts, INPs and their immediate neural progeny- GMCs, detected by a specific antibody generated against Pfdn2 full length and a transgenic Pfdn2 with a Venus tag at the C-terminus. In addition, Pfdn2 expression under the tubulin-Gal4 fully rescued the lethality of both pfdn2Δ10 and pfdn2Δ17 mutants. Pfdn2 protein was undetectable in pfdn2Δ10 zygotic mutants, further supporting that it is a null allele. Both type I and type II MARCM (Mosaic Analysis with Repressible Cell Marker) clones of pfdn2Δ10 generated excess neuroblasts. These phenotypes were slightly weaker than pfdn2Δ10 zygotic mutants, likely due to residual Pfdn2 protein in the clones. These data indicate that Pfdn2 is required in both type I and type II neuroblast lineages to prevent the formation of ectopic neuroblasts (Zhang, 2016).
This study has identified an unexpected synergism between Prefoldin and Pins in suppressing neuroblasts overgrowth. Barious subunits of Prefoldin complex are implicated in asymmetric division of neuroblasts, especially during asymmetric protein segregation at telophase. It is known that depletion of Pins results in the formation of smaller larval brains, despite partial loss of neuroblasts polarity. Interestingly, co-depletion of Pfdn2 and Pins results in severe neuroblasts overgrowth, while Pfdn2 depletion alone only causes mild brain overgrowth. This phenotype is contributed by a combination of loss of neuroblast polarity, defects of asymmetric division of INPs, as well as INP dedifferentiation. Knocking down tubulins in pins mutant background mimics the co-depletion of Prefoldin and Pins, suggesting that tubulin stability appears to be critical for the suppression of neuroblast overgrowth in the absence of Pins function. The data also suggest that Prefoldin function and tubulin stability in INPs are important to suppress their dedifferentiation back into neuroblasts (Zhang, 2016).
How microtubules induce cortical polarity is poorly understood in Drosophila neuroblasts. Previously, one report showed that kinesin Khc-73, which localized at the plus end of astral microtubules, and Discs large (Dlg) induced cortical polarization of Pins/Gαi in neuroblasts. However, microtubules are considered not essential for neuroblast polarity. This study shows that Drosophila Prefoldin regulates asymmetric division of both neuroblasts and INPs through tubulins, suggesting an important role of microtubules in neuroblast polarity. The essential role of microtubules directly regulating cell polarity is found in various systems. During C. elegans meiosis, a microtubule-organizing center is necessary and sufficient for the establishment of the anterior-posterior polarity. In the fission yeast Schizosaccharomyces pombe, interphase microtubules directly regulate cell polarity through proteins such as tea1p. In mammalian airway cilia, microtubules are required for asymmetric localization of planer cell polarity proteins (Zhang, 2016).
This study shows that the role of Drosophila Prefoldin complex in regulating asymmetric division is very likely dependent on microtubules. This is consistent with the known essential role of Prefoldin for maintaining tubulin levels in various organisms such as yeast, C. elegans, plants and mammals. In yeast, Gim (Prefoldin) null mutants become super-sensitive to the microtubule-depolymerizing drug benomyl as a result of a reduced level of α-tubulin. In the absence of Prefoldin, the function of the chaperone pathway is damaged and unable to fold sufficient amount of tubulins for normal yeast growth. In C. elegans, reducing Prefoldin function causes defects in cell division presumably due to the reduction of tubulin levels and microtubule growth rate. Genetic analysis of mammalian Prefoldin also suggests that cytoskeletal proteins like actin and tubulin make up the major substrate of Prefoldin in mammals. These studies in different organisms together suggest that Prefoldin complex plays a conserved central role in tubulin folding (Zhang, 2016).
'Telophase rescue', a term refers to the phenomenon that protein mis-localization at metaphase is completely restored at telophase, is observed in many mutants that affect neuroblast asymmetric division. However, both apical and basal proteins are still mis-segregated in pfdn2 and mgr mutants, suggesting that 'telophase rescue' is defective in these mutants. Telophase rescue is regulated by TNF receptor-associated factor (DTRAF1), which binds to Baz and acts downstream of Egr/TNF. Telophase rescue also depends on Worniu/Escargot/Snail family proteins and a microtubule-dependent Khc-73/Dlg pathway. Pins did not form a protein complex with Mgr, α-tubulin or β-tubulin in co-immunoprecipitation assay. Given that Dlg is a Pins-interacting protein, Prefoldin appears to function in a different pathway with Dlg or Khc-73 during asymmetric division (Zhang, 2016).
Recently, merry-go-round (mgr), encoding Prefoldin 3 (Pfdn3)/VBP1/Gim2 subunit, was reported to regulate spindle assembly. Loss of mgr led to formation of monopolar mitotic spindles and loss of centrosomes because of improper folding and destabilization of tubulins. The current analysis on Pfdn2 indicates that pfdn2 mutants displayed similar spindle and centrosome abnormalities. In addition, the incorrectly folded tubulin due to loss of mgr may be eliminated by Drosophila von Hippel Lindau protein (Vhl), an E3 ubiquitin-protein ligase. Interestingly, the data suggest that Prefoldin has a tumor-suppressor like function in preventing neuroblast overgrowth. However, Drosophila Vhl is not important for brain tumor suppression, as its loss-of-function neither affects number of neuroblasts nor suppresses overgrowth observed in pfdn2 RNAi or mgr RNAi (Zhang, 2016).
This study shows a novel synergism between Prefoldin and Pins in suppressing dedifferentiation of INPs back into neuroblasts. Prefoldin and Pins apparently suppress dedifferentiation through regulating tubulin levels. It is likely that appropriate tubulin levels in INPs are important for their differentiation, while reducing tubulin levels can increase the risk of INP dedifferentiation. Currently, several cell fate determinants such as Brat, Numb and the SWI/SNF chromatin remodeling complex with its cofactors Erm and Hdac3 are critical to suppress INP dedifferentiation back into neuroblast. It is currently unknown whether or how Prefoldin/Pins are linked to these known suppressors of dedifferentiation. It is possible that symmetric division of INPs causes reduced levels of Brat and Numb in these abnormal INP daughters, leading to their dedifferentiation. Alternatively, Prefoldin might regulate transcription of genes within INPs to suppress dedifferentiation. It was reported that the human homolog of Pfdn5, MM-1, has a role in transcriptional regulation by binding to the E-box domain of c-Myc and represses E-box-dependent transcriptional activity. Interestingly, Prefoldin Subunit 5 gene is deleted in Canine mammary tumors, suggesting that it may be a tumor suppressor gene. This study has revealed a novel mechanism by which Prefoldin and Pins function through tubulin stability to suppress stem cell overgrowth. It is expected to contribute to the understanding of mammalian/human Prefoldin function in tumorigenesis (Zhang, 2016).
forward to beta Tubulin 56D Protein interactions part 2/2
betaTubulin56D (ß1 tubulin):
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
| References
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