Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles.

Intraflagellar transport (IFT) is a motility in which particles composed of at least 17 polypeptides move underneath the flagellar membrane. Anterograde (outward) and retrograde (inward) movements of these IFT particles are mediated by FLA10 kinesin-II and cytoplasmic dynein DHC1b, respectively. Mutations affecting IFT particle polypeptides or motors result in the inability to assemble flagella. IFT particles and the motors moving them are located principally around the basal bodies as well as in the flagella. Here, we clone the cDNA encoding one of the IFT particle proteins, IFT52, and show by immunofluorescence that while some IFT52 is in the flagella, the majority is found in two horseshoe-shaped rings around the basal bodies. Immunoelectron microscopy indicates that IFT52 is associated with the periphery of the transitional fibers, which extend from the distal portion of the basal body to the cell membrane and demarcate the entrance to the flagellar compartment. This localization suggests that the transitional fibers form a docking complex for the IFT particles destined for the flagellum. Finally, the flagellaless mutant bld1 completely lacks IFT52 due to a deletion in the gene encoding IFT52.

Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella.

Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.

Kinesin-II, the heteromeric kinesin.

The kinesins constitute a large family of motor proteins which are responsible for the distribution of numerous organelles, vesicles and macromolecular complexes throughout the cell. One class of these molecular motors, kinesin-II, is unique in that these proteins are typically found as heterotrimeric complexes containing two different, though related, kinesin-like motor subunits, and a single nonmotor subunit. The heteromeric nature of these kinesins appears to have resulted in a class of combinatorial kinesins which can 'mix and match' different motor subunits. Another novel feature of these motors is that the activities of several kinesin-II representatives are essential in the assembly of motile and nonmotile cilia, a role not attributed to any other kinesin. This review presents a brief overview of the structure and biological functions of kinesin-II, the heteromeric kinesin.

Purification of novel kinesins from embryonic systems.

Several kinesin holoenzymes, including the heterotrimeric kinesin-II and bipolar KLP61F complexes described here, are being purified in our laboratory using microtubule affinity precipitation and conventional biochemical fractionation procedures. These protocols have been optimized by using pan-kinesin peptide antibodies and subunit-specific antibodies to monitor the enrichment of kinesin-related polypeptides in particular fractions by immunoblotting. Protein purification represents the most direct route available for determining the oligomeric state and subunit composition of a kinesin holoenzyme, for identifying tightly associated accessory subunits such as SpKAP115, and for determining the molecular architecture and functional properties of native kinesin motors. Protein purification methods therefore represent an important complementary approach to molecular genetic approaches that are being pursued in many other laboratories.

DARPP-32: regulator of the efficacy of dopaminergic neurotransmission.

Dopaminergic neurons exert a major modulatory effect on the forebrain. Dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein (32 kilodaltons) (DARPP-32), which is enriched in all neurons that receive a dopaminergic input, is converted in response to dopamine into a potent protein phosphatase inhibitor. Mice generated to contain a targeted disruption of the DARPP-32 gene showed profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication. The results show that DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission.

Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons.

We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979-992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia.

The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm.

We have utilized immunoblotting and light microscopic immunofluorescent staining methods to examine the expression and localization of sea urchin kinesin-II, a heterotrimeric plus end-directed microtubule motor protein (previously referred to as KRP(85/95)), in sea urchin and sand dollar sperm. We demonstrate the presence of the 85 K and 115 K subunits of kinesin-II in sperm and localize these proteins to the sperm flagella and midpiece. The kinesin-II localization pattern is punctate and discontinuous, and in the flagella it is quite distinct from the continuous labeling present in sperm labeled with anti-flagellar dynein. The kinesin-II staining is largely insensitive to prefixation detergent extraction, suggesting that it is not associated with membranous elements in the sperm. In the midpiece the kinesin-II staining is similar to the pattern present in sperm labeled with an anti-centrosomal antibody. To our knowledge, this is the first localization of kinesin-like proteins in mature sperm and corroborates the recent identification and localization of kinesin-like proteins in the flagella and basal body of the unicellular green alga Chlamydomonas. We hypothesize that kinesin-II in the sperm may play functional roles in intraflagellar transport and/or the formation of flagella during spermatogenesis.

Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP85/95) complex.

The heterotrimeric kinesin-II holoenzyme purified from sea urchin (Strongylocentrotus purpuratus) eggs is assembled from two heterodimerized kinesin-related motor subunits of known sequence, together with a third, previously uncharacterized 115-kD subunit, SpKAP115. Using monospecific anti-SpKAP115 antibodies we have accomplished the molecular cloning and sequencing of the SpKAP115 subunit. The deduced sequence predicts a globular 95-kD non-motor "accessory" polypeptide rich in alpha-helical segments that are generally not predicted to form coiled coils. Electron microscopy of individual rotary shadowed kinesin-II holoenzymes also suggests that SpKAP115 is globular, with a somewhat asymmetric morphology. Moreover, the SpKAP115 subunit lies at one end of the 51-nm-long kinesin-II complex, being separated from the two presumptive motor domains by a approximately 26-nm-long rod, in a manner similar to the light chains (KLCs) of kinesin itself. This indicates that SpKAP115 and the KLCs may have analogous functions, yet SpKAP115 does not display significant sequence similarity with the KLCs. The results show that kinesin and kinesin-II are assembled from highly divergent accessory polypeptides together with kinesin related motor subunits (KRPs) containing conserved motor domains linked to divergent tails. Despite the lack of sequence conservation outside the motor domains, there is striking conservation of the ultrastructure of the kinesin and kinesin-II holoenzymes.

A bipolar kinesin.

Chromosome segregation during mitosis depends on the action of the mitotic spindle, a self-organizing, bipolar protein machine which uses microtubules (MTs) and their associated motors. 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. Here we report that KRP130, a homotetrameric BimC-related kinesin purified from Drosophila melanogaster embryos, 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. We do not know of any other MT motors that have a bipolar structure.

Immunolocalization of the heterotrimeric kinesin-related protein KRP(85/95) in the mitotic apparatus of sea urchin embryos.

We have used monoclonal antibodies to perform confocal light microscopic immunolocalization of KRP(85/95), a heterotrimeric plus-end-directed microtubule motor protein, in dividing cells of sea urchin embryos. Embryos were stained during the first division cycle, and dissociated blastomeres were stained at the 32- to 64-cell stages. Double labeling of the dividing cells with anti-tubulin and anti-KRP(85/95) showed a clear concentration of the motor protein in the mitotic apparatus; KRP(85/95) appeared to associate with pericentriolar regions during prophase, with kinetochore-to-pole microtubules during metaphase, and, in a striking fashion, with the spindle interzone during anaphase. KRP(85/95) began to accumulate in the interzone immediately following chromosome separation and the area of concentration expanded with the lengthening of the interzonal region during anaphase. During telophase KRP(85/95) appeared to disperse with the establishment of the cleavage furrow and did not concentrate in the midbody. KRP(85/95) staining in the mitotic apparatus was punctate and detergent-sensitive, suggesting an association with membranous vesicles, but unlike kinesin, KRP(85/95) did not appear to codistribute with calsequestrin-containing endoplasmic reticulum. Finally, KRP(85/95) appears to be present in dividing blastomeres up to at least the blastula stage, but, unlike kinesin, it is not expressed in terminally differentiated, nonmitotic coelomocytes of the adult animal. These results suggest that the expression and targeting of KRP(85/95) and kinesin differ and that KRP(85/95) may play a role in vesicle transport during embryonic cell division.

Structural variations among the kinesins.

Members of the kinesin family of motor proteins are assembled from kinesin-related polypeptides that share conserved 'motor' domains linked to diverse 'tail' domains. Recent work suggests that tail diversity underlies the differences in quaternary structure observed among native kinesin holoenzymes.

Purification of kinesin-related protein complexes from eggs and embryos.

We have developed a biochemical screen for the identification of kinesin-related proteins (KRPs) in their natural host cells and the subsequent purification of these KRPs as native, functional multimeric complexes. The screen involves immunoblotting with pan-kinesin peptide antibodies that recognize several presumptive KRPs in cytosolic extracts; the antibodies have been used so far to monitor the purification of two bona fide kinesin-related motor protein complexes. These two KRPs were purified via AMPPNP-induced microtubule affinity binding, ATP-induced elution from AMPPNP microtubules, gel filtration fractionation, and sucrose density gradient centrifugation. KRP(85/95) from sea urchin (Strongylocentrotus purpuratus) eggs behaves as a heterotrimeric complex of 85-, 95-, and 115-kDa subunits that moves toward the plus ends of microtubule tracks at approximately 0.4 micron/s. KRP(130) from fruitfly (Drosophila melanogaster) embryos behaves as a homotetrameric complex of four 130-kDa subunits that moves toward the plus ends of microtubule tracks at approximately 0.04 micron/s. To our knowledge, KRP(85/95) and KRP(130) are the only KRPs to have been purified from native tissue as functional multimeric motor complexes.

A "slow" homotetrameric kinesin-related motor protein purified from Drosophila embryos.

Pan-kinesin peptide antibodies (Cole, D. G., Cande, W. Z., Baskin, R. J., Skoufias, D. A., Hogan, C. J., and Scholey, J. M. (1992) J. Cell Sci. 101, 291-301; Sawin, K. E., Mitchinson, T. J., and Wordeman, L. G. (1992) J. Cell Sci. 101, 303-313) were used to identify and isolate kinesin-related proteins (KRPs) from Drosophila melanogaster embryonic cytosol. These KRPs cosedimented with microtubules (MTs) polymerized from cytosol treated with AMP-PNP (adenyl-5'-yl imidodiphosphate), and one of them, KRP130, was further purified from ATP eluates of the embryonic MTs. Purified KRP130 behaves as a homotetrameric complex composed of four 130-kDa polypeptide subunits which displays a "slow" plus-end directed motor activity capable of moving single MTs at 0.04 +/- 0.01 microns/s. The 130-kDa subunit of KRP130 was tested for reactivity with monoclonal and polyclonal antibodies that are specific for various members of the kinesin superfamily. Results indicate that the KRP130 subunit is related to Xenopus Eg5 (Sawin, K. E., Le Guellec, K. L., Philippe, M., Mitchinson, T. J. (1992) Nature 359, 540-543), 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.

6-Hydroxydopamine lesions of rat substantia nigra up-regulate dopamine-induced phosphorylation of the cAMP-response element-binding protein in striatal neurons.

Destruction of the substantia nigra produces striatal D1 dopamine receptor supersensitivity without increasing receptor number or affinity, thus implicating postreceptor mechanisms. The nature of these mechanisms is unknown. Increased striatal c-fos expression ipsilateral to a unilateral lesion of the substantia nigra in rats treated with appropriate dopamine agonists provides a cellular marker of D1 receptor supersensitivity. D1 receptors are positively linked to adenylate cyclase and therefore to cAMP-dependent protein kinase. Because expression of the c-fos gene in response to cAMP- and Ca2+/calmodulin-regulated protein kinases depends on phosphorylation of cAMP-response element-binding protein (CREB) at Ser-133, we examined CREB phosphorylation after dopaminergic stimulation in cultured striatal neurons and in the striatum of rats after unilateral 6-hydroxydopamine ablation of the substantia nigra. Using an antiserum specific for CREB phosphorylated at Ser-133, we found that dopamine increases CREB phosphorylation in cultured striatal neurons. This effect was blocked by a D1 antagonist. L-Dopa produced marked CREB phosphorylation in striatal neurons in rats ipsilateral, but not contralateral, to a 6-hydroxydopamine lesion. This response was blocked by a D1 antagonist, but not a D2 antagonist, and was reproduced by a D1 agonist, but not a D2 agonist. These findings are consistent with the hypothesis that D1 receptor supersensitivity is associated with upregulated activity of cAMP-dependent or Ca2+/calmodulin-dependent protein kinases, or both, following dopamine denervation of striatal neurons.

Reserpine increases Fos activity in the rat basal ganglia via a quinpirole-sensitive mechanism.

Expression of the immediate early gene c-fos increases acutely following neuronal depolarization. c-fos and Fos protein have been widely used to investigate basal ganglia responses to changes in dopaminergic neurotransmission. Increased dopaminergic input to D1 receptors increases Fos synthesis in striatal neurons. The role of D2 receptors in regulating Fos activity has been more difficult to establish. Because dopamine is believed to excite striatal neurons via D1 receptors and inhibit them via D2 receptors, we hypothesized that acute dopamine depletion would increase Fos activity in basal ganglia circuits normally inhibited by dopaminergic input to D2 receptors. Rats were perfused after a single dose of the dopamine-depleting drug reserpine. The brains of rats perfused 3 h after reserpine displayed numerous Fos-like immunoreactive nuclei in the striatum, entopeduncular nucleus, nucleus accumbens shell, and ventral pallidum, and sparse Fos-like immunoreactive nuclei in the globus pallidus and nucleus accumbens core. Few or no Fos-like immunoreactive nuclei were seen following perfusion 30 min, 60 min, and 24 h after reserpine. In the 3-h paradigm, pretreatment with the selective D1 antagonist SCH 23390 did not change the pattern of Fos-like immunoreactivity; pretreatment with the selective D2 agonist quinpirole completely blocked increased Fos synthesis. Acute dopamine depletion, therefore, increases Fos activity in the basal ganglia by disinhibiting D2 circuits. These results support the parallel pathway model of basal ganglia function, and show that Fos can be used to investigate the role of D2 receptors in striatal function. The findings suggest anatomic correlates for the clinical effects of acute dopamine depletion in drug therapy and advanced Parkinson's disease in humans.

Therapy for progressive supranuclear palsy: past and future.

Dysfunction of multiple brain systems in progressive supranuclear palsy (PSP) has complicated attempts to treat the disease. Neurotransmitter replacement strategies targeting the dopaminergic, cholinergic, and serotonergic systems have been unsuccessful. In order to bypass the degenerated cortico-striato-pallidal loop, we administered the adrenergic agonist idazoxan (IDA) to treat PSP in two randomized double-blind, placebo controlled, crossover studies. Approximately one half of patients enrolled in these studies showed statistically significant improvement in balance and manual dexterity while taking IDA compared to placebo. These results suggest that new therapies that target structures outside of the basal ganglia may be useful for symptomatic treatment of PSP. Applying this strategy and developing treatments that arrest or reverse clinical deterioration in PSP will require improved understanding of the process underlying the illness.

The carboxyl-terminal domain of kinesin heavy chain is important for membrane binding.

Sea urchin kinesin is a plus end-directed microtubule-based motor consisting of two heavy chains and two light chains and is proposed to be responsible (a) for the transport of membranous organelles along microtubules in sea urchin mitotic spindles (Wright, B. D., Henson, J. H., Wedaman, K. P., Willy, P. J., Morand, J. N., and Scholey, J. M. (1991) J. Cell Biol. 113, 817-833) and (b) for the radial dispersion of endoplasmic reticulum and endosomal membranes in non-mitotic cultured coelomocytes (Henson, J. H., Nesbitt, D., Wright, B. D., and Scholey, J. M. (1992) J. Cell Sci. 103, 309-320). We report here that sea urchin kinesin is indeed able to bind in a concentration-dependent and saturable manner to microsomal membranes isolated from sea urchin eggs in the presence of MgATP. The kinesin light chains may not be essential for membrane binding since kinesin containing negligible amounts of light chains binds as well as kinesin containing stoichiometric amounts of light chains. Finally, we propose that kinesin binds to membranes with the carboxyl-terminal domain of the heavy chain (amino acid residues 858-1031) since the bacterially expressed and then isolated stalk-tail fragment of kinesin heavy chain, in contrast to the stalk fragment, is able (a) to bind membranes in a concentration-dependent and saturable manner and (b) to compete with native kinesin for membrane binding. Our results support the hypothesis that the carboxyl-terminal domains of the heavy chains attach kinesin molecules to their membranous cargo in mitotic and interphase sea urchin cells.