Desai A et al. (1999) Cell "Kin I kinesins are microtubule-destabilizing enzymes."

Mitchison TJ, Verma S, Walczak CE, Desai A

[Cell, 1999]

Using in vitro assays with purified proteins, we show that XKCM1 and XKIF2, two distinct members of the internal catalytic domain (Kin I) kinesin subfamily, catalytically destabilize microtubules using a novel mechanism. Both XKCM1 and XKIF2 influence microtubule stability by targeting directly to microtubule ends where they induce a destabilizing conformational change. ATP hydrolysis recycles XKCM1/XKIF2 for multiple rounds of action by dissociating a XKCM1/ XKIF2-tubulin dimer complex released upon microtubule depolymerization. These results establish Kin I kinesins as microtubule-destabilizing enzymes, distinguish them mechanistically from kinesin superfamily members that use ATP hydrolysis to translocate along microtubules, and have important implications for the regulation of microtubule dynamics and for the intracellular functions and evolution of the kinesin superfamily.

Desai C et al. (1984) Worm Breeder's Gazette "the HSNs appear to be serotonergic."

Desai C, Horvitz HR, McIntire SL

[Worm Breeder's Gazette, 1984]

The two HSNs ('hermaphrodite-specific neurons') innervate the vulval muscles and are necessary for normal egg-laying by the C. elegans hermaphrodite. Wild-type hermaphrodites are stimulated to lay eggs by exogenous serotonin or imipramine. Hermaphrodites lacking the HSNs are egg-laying defective but are stimulated to lay eggs by exogenous serotonin (Trent et. al., Genetics 104: 619, 1983). In contrast, imipramine does not stimulate egg-laying by HSN-defective hermaphrodites. In other organisms, imipramine is believed to potentiate endogenous serotonin (specifically, imipramine is thought to inhibit the uptake of serotonin into presynaptic terminals and thus to increase the amount of serotonin in the synaptic cleft). The simplest interpretation of these data is that the HSNs are serotonergic: exogenous serotonin bypasses the need for input from the HSNs; imipramine fails to stimulate egg-laying by an HSN-defective animal because there is no serotonergic input from the HSNs to potentiate. Serotonin has been localized histochemically in C. elegans using the technique of formaldehyde-induced fluorescence (FIF) (Horvitz et al., Science 216: 1012, 1982). With this technique only the two pharyngeal NSMs neurosecretory-motor neurons') could be seen to be serotonergic. He have now used anti-serotonin antisera to attempt to confirm the serotonergic nature of the NSMs and to identify other serotonergic cells. After fixation in formaldehyde, animals are digested in elastase and/or collagenase to permeabilize the cuticle to antibodies. The primary rabbit anti-serotonin antibody is visualized using a rhodamine-conjugated goat anti-rabbit antibody. This antibody protocol has proved to be significantly more sensitive than FIF: NSM cell bodies as well as processes can be seen (FIF shows only the processes in animals that have not been exposed to exogenous serotonin) , and a variety of other cells are stained. These new, possibly serotonergic cells include about six cells in the head, two cells in the pharynx, six bright and four faint cells in the male ventral cord, four or five cells in the male tail, and the HSN cell bodies and processes in the hermaphrodite. We have isolated 45 mutants that appear to be functionally HSN- defective by the criteria described above (see Desai et al., C. elegans Newsletter 8: 26, 1983). Some of these mutants lack HSNs completely, some have variably absent or misplaced HSNs, and some have morphologically normal HSNs as visualized with Nomarski optics. He have now utilized the anti-serotonin antibody to subdivide this last category into two groups: n1126 and n1077 (which represent two genes) do not stain for serotonin, whereas n995, n1068 and n1082 (which represent three genes) have normally staining HSNs. Strikingly, the mutants that lack serotonin staining in the HSNs contain serotonin in at least most of the other putative serotonergic cells. We have also discovered that n997, one of the mutants previously described as lacking HSNs, displays a pair of serotonergic cells variably positioned between the vulva and the tail. This mutant appears likely to be defective in HSN migrations, which occur embryonically as these cells move from the tail to the vulva (Sulston et al., Devel. Biol. 100: 64, 1983). No other defects have been observed in this mutant, suggesting that it defines a gene that may be involved specifically in HSN migration. It is interesting that these displaced HSNs become serotonergic and send processes into the ventral cord to the nerve ring, although presumably functional synapses onto the vulval muscles are not formed.

Desai C et al. (1984) Worm Breeder's Gazette "more about HSN-defective mutants."

Horvitz HR, Desai C

[Worm Breeder's Gazette, 1984]

We have isolated 45 C. elegans egg-laying defective mutants that appear to have functionally impaired HSNs (see Newsletter Vol. 8 No. 1). (The HSNs are two motorneurons that innervate the vulval muscles.) These mutants define at least 14 genes, of which we have now mapped 11 (see Map on the next page). In the last Newsletter (Vol. 8 No. 2) we reported that in the wild type the HSNs are stained by anti- serotonin antisera, and we described the phenotypes of some of these HSN-defective mutants as observed after staining with anti-serotonin antisera. We have now examined the appearance of the HSNs in mutants representing all 14 of these genes using indirect immunofluorescence to visualize serotonin and Nomarski optics to visualize cell bodies ( see Table on the next page; an * in the table indicates a mutant allele studied by microscopy). These HSN-defective mutants can be divided into five categories. Mutants in Category A lack HSNs by both criteria; mutations in three of the five genes of this category appear to cause HSNs to undergo programmed cell death, as these mutations are suppressed by mutations in the genes ced-3 or ced-4 (which block the onset of programmed cell deaths; see Ellis et al., this Newsletter). Mutants in Category B appear to generate abnormal HSNs, which can be absent or variably mispositioned and which when present invariably lack serotonin. The mutant egl-46(n1127), which defines Category C, has HSN cell bodies in almost normal positions but lacks serotonin in the HSNs. (This mutant contains serotonin in the other normally serotonin-positive neurons that are visualized after staining with the anti-serotonin anti-sera.) It has not been unambiguously demonstrated that the cells considered to be misplaced HSNs in Categories B and C mutants are indeed HSNs. The mutant egl-43(n997), which defines Category D, appears to be defective in the migration of the HSNs. (HSN migration is a normal aspect of embryonic development.) As seen with Nomarski optics, egl-43 animals lack HSNs in their normal positions; serotonin-staining reveals ectopic serotonin-positive cells variably located along the path of HSN migration (from the postanal region to near the position of the vulva). A double mutant containing egl-43(n997) and the Category A mutation egl-41(n1069) lacks these ectopic serotonin- positive cells, indicating that they are mispositioned HSNs. The misplaced putative HSNs of egl-43 animals have aberrant processes: some run abnormally long distances along the ventral cord into the nerve ring (normal HSNs run from the region of the vulva along the ventral cord into the nerve ring), others are bipolar, and others send a process posteriorly to the tip of the tail or anteriorly via a sublateral route. The putative HSNs of egl-43 animals seem to migrate slightly farther on average at 20 C or 25 C an they do at 15 C. Category E mutants have HSNs that appear normal as visualized with both Nomarski and fluorescence microscopy. [See figure 1]

Desai C et al. (1987) International C. elegans Meeting "genes involved in the development of the HSN neurons."

McIntire SL, Garriga G, Desai C, Horvitz HR, Thomson JN

[International C. elegans Meeting, 1987]

Desai C et al. (1985) International C. elegans Meeting "mutation that impair the functioning of the HSN neurons."

Horvitz HR, Desai C

[International C. elegans Meeting, 1985]

Desai A et al. (2003) Genes Dev "KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C. elegans."

Oegema K, Shevchenko A, Desai A, Muller-Reichert T, Rybina S, Hyman A

[Genes Dev, 2003]

Segregation of the replicated genome during cell division requires kinetochores, mechanochemical organelles that assemble on mitotic chromosomes to connect them to spindle microtubules. CENP-A, a histone H3 variant, and CENP-C, a conserved structural protein, form the DNA-proximal foundation for kinetochore assembly. Using RNA interference-based genomics in Caenorhabditis elegans, we identified KNL-1, a novel kinetochore protein whose depletion, like that of CeCENP-A or CeCENP-C, leads to a "kinetochore-null" phenotype. KNL-1 is downstream of CeCENP-A and CeCENP-C in a linear assembly hierarchy. In embryonic extracts, KNL-1 exhibits substoichiometric interactions with CeCENP-C and forms a near-stoichiometric complex with CeNDC-80 and HIM-10, the C. elegans homologs of Ndc80p/HEC1p and Nuf2p-two widely conserved outer kinetochore components. However, CeNDC-80 and HIM-10 are not functionally equivalent to KNL-1 because their inhibition, although preventing formation of a mechanically stable kinetochore-microtubule interface and causing chromosome missegregation, does not result in a kinetochore-null phenotype. The greater functional importance of KNL-1 may be due to its requirement for targeting multiple components of the outer kinetochore, including CeNDC-80 and HIM-10. Thus, KNL-1 plays a central role in translating the initiation of kinetochore assembly by CeCENP-A and CeCENP-C into the formation of a functional

Desai C et al. (1983) Worm Breeder's Gazette "egg-laying defective mutants with abnormal HSN function."

Desai C, Horvitz HR

[Worm Breeder's Gazette, 1983]

The two 'hermaphrodite-specific neurons' (HSNs) of C. elegans are crucial for wild-type egg-laying. The unique pharmacological characteristics of hermaphrodites lacking the HSNs have provided us with an opportunity to study the genetics of the development and functioning of this single class of neuron: whereas wild-type hermaphrodites are stimulated to lay eggs by both exogenously added imipramine and serotonin, worms in which the HSNs have been eliminated (either by laser microbeam ablation or by mutation, as in unc-86 or egl-1 animals) are responsive only to serotonin. Such HSN worms retain eggs but eventually release most or all of their progeny. In the dissecting microscope HSN animals are obviously swollen and are whitish due to the presence in the uterus of late stage eggs and, sometimes, hatched larvae. The progeny of 40,000 F1's from EMS-mutagenized parents were screened for animals with this swollen phenotype. Egl candidates were picked and their progeny tested with serotonin and imipramine. Forty- five mutants sensitive to serotonin and resistant to imipramine were isolated. These mutations define at least 16 genes, including five that were previously known: tra-2 II, unc-86 III, egl-5 III, egl-1 V and egl-10 V. New loci are defined by (n1086) n995,n996) II; (n997) II; (n998,n1079,n1080,n1087) II; (n999) n1077) V (because these mutations are dominant they are only tentatively assigned to the same locus); (n1081,n1082) V; (n1108) V; (n1075,n1076) V; and (n1107) X. Mutants defective in eight of these loci have normal HSN cell bodies as observed with Nomarski optics. Mutants defective in three other loci have variably missing and often abnormal HSNs, while mutants defective in the remaining five loci completely lack HSN cell bodies. The HSNs in mutants defective in two of these last five loci --egl-1 and egl(n1069) appear to lack HSNs because these cells undergo programmed cell death. All seven mutations defining these two genes regain both wild-type egg-laying behavior and HSN cell bodies in double mutants with ced-3, which prevents programmed cell deaths. Mutations in both loci are dominant and of variable expressivity. These two genes map within 1 m. u. of each other on linkage group V, but are separated by him-5. All alleles of egl-1 are heat-sensitive, whereas all alleles of egl(n1069) are cold-sensitive. Mutations in these two genes interact, as the double heterozygote is more Egl than expected from the penetrance of the single heterozygotes alone. Genes that control HSN cell deaths may be involved in the expression of sexual phenotype. For example, the HSNs in egl-1 and egl(n1069) may undergo the normally male specific programmed cell death in the hermaphrodite. In addition, alleles of the general sex determination loci may result in a partial sexual transformation that leads to the deaths of the HSNs in hermaphrodites; both her-1 and tra-2 alleles of this class have been previously identified (Trent, Tsung and Horvitz, Genetics 104, 619, 1983). We have recently isolated a new tra-2 allele, n1106, that is also of this class. n1106 results in a very low fraction of intersex animals, a high fraction of Egl hermaphrodites and a few wild-type hermaphrodites. Most n1106 animals lack both HSN cell bodies, and most of the rest lack one. As expected, the HSNs are restored and the egg-laying defect of tra-2(n1106) is largely suppressed by ced-3, whereas other phenotypic indications of maleness are not.

Desai T et al. (2004) Physica Medica "Investigation of Caenorhabditis elegans using soft X-ray contact microscopy."

Mocek T, Krousky E, Batani D, Kralikova B, Pfeifer M, Juha L, Donin CLL, Bernardiello A, Zullini A, Poletti G, Prag A, Desai T, Kadlec C, Renner O, Skala J, Orsini F, Ullschmied J, Cotelli F

[Physica Medica, 2004]

Soft X-ray Contact Microscopy (sxcm) experiments have been performed on the nematode Caenorhabditis elegans using the PragueAsterix Iodine Laser System (pals). Pulsed X-rays were generated using gold and molybdenum targets with laser intensities I 1014W/cm2 to record the contact images of C. elegans on pmma photoresists. Results were analyzed using an atomic force microscopeoperating in constant force mode. Our results show some characteristic features of the C. elegans and the suitability of the sxcmtechnique for the study of multi-cellular specimens.

Desai C et al. (1988) Nature "A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons."

Desai C, Horvitz HR, Garriga G, McIntire SL

[Nature, 1988]

Thirty-five genes define a pathway for the development of the hermaphrodite-specific neurons (HSNs) in Caenorhabditis elegans. Some of these genes affect only one HSN trait, demonstrating that HSN migration, axonal outgrowth and serotonin expression are mutually independent events in HSN development; others, some of which are regulatory, affect multiple HSN traits. Nearly all are pleiotropic, revealing that the genes specifying HSN development also function in the development of other cell types.

Desai C et al. (1985) Worm Breeder's Gazette "the phenotype of the HSNs in egl-44 and egl-46 animals may depend on interaction with their targets."

Desai C, Horvitz HR

[Worm Breeder's Gazette, 1985]

We have previously described 45 mutations that disrupt the functioning of the HSN motorneurons, which drive egglaying. Of these mutations, seven that define the two genes egl-44 and egl-46 display low levels of serotonin in the HSNs as visualized by indirect immunoflourescent staining. The other serotonergic cells (the NSMs and six or seven additional neurons in the head and pharynx) appear to have normal levels of serotonin. While constructing and characterizing strains containing pairs of mutations affecting the HSNs, we noticed that the HSNs in egl-43: egl-46 animals have increased levels of anti-serotonin staining compared to the levels seen in egl-44 and egl-46 mutants alone. The HSNs in egl-43 mutants fail to migrate from their birthplaces in the tail to their wild-type positions just posterior to the midpoint of the gonad. This result led us to theorize that the lower antiserotonin staining in egl-44 and egl-46 HSNs may be dependent on synapse formation between the HSN processes and their targets, the vulval muscles. To further test this hypothesis, we constructed four more double mutant strains, designed to disrupt these synapses. These double mutant strains are: egl-44: egl-44; egl-46: egl-31. The egl-15 mutation results in the incomplete migrations of the sex myoblasts, the ancestors of the vulval muscle cells. The egl-31 mutation results in abnormalities in the M cell divisions that generate the sex myoblasts. The level of anti-serotonin staining in the HSNs of each of these four strains is enhanced, compared to that seen in egl-44 and egl-46 alone. All six of the double mutant strains described above either remove the targets from the HSNs or the HSNs from their targets. Thus, the low level of anti-serotonin staining found in the HSNs of egl-44 and egl-46 animals seems to depend on HSN-vulval muscle synapse formation. This reduced anti-serotonin staining could be due to synaptic leakage of serotonin, faulty reuptake, or some other defect.