Badrinath AS et al. (1998) Worm Breeder's Gazette "Cloning and Characterization of the Cytokinesis mutant stu-4."

White JG, Badrinath AS

[Worm Breeder's Gazette, 1998]

We are characterizing the temperature-sensitive mutant stu-4 (e2406) (originally isolated by David Livingstone) which maps to -4.42 on the left arm of LGI. When gravid adults are shifted to the non-permissive temperature 100% of the embryos die due to maternal effect lethality. We examined the cause of this lethality using 4-D time-lapse (Nomarski) microscopy. The polar bodies fail to be extruded resulting in several ooctye !pronuclei! migrating toward the posteriorly located sperm pronucleus where they generally meet at the center of the embryo. Spindle formation, and reformation of the nuclei following chromosome segregation appears to be normal, but at no time point is there any sign of furrowing at the cortex. This results in a multi-nucleated single celled embryo. When L2 or L3 larval stage e2406 animals are shifted to the non permissive temperature 100% of them become sterile and exhibit an uncoordinated phenotype to a lesser extent. These defects are presumably due to failures in cell divisions in the post-embryonic lineages giving rise to the gonad and ventral nerve cord respectively. At an intermediate temperature of 20 degrees only 7 per cent of the homozygotes shifted as L2 or L3 larvae were fertile. We were able to rescue the post-embryonic defects with a small group of cosmids containing K03E5, W05F2 and W07C11. Up to 60% of the rolling homozygotes shifted to 20 degrees as L2 or L3 larvae becoming fertile adults. Of these three cosmids only K03E5 had been sequenced by the genome consortium, and contains 5 predicted genes. One of them K03E5.cand3 codes for a protein with strong homology to calponins which are actin-binding proteins that regulate acto-myosin contraction in a calcium-dependent manner in smooth muscles. When this gene!s function was perturbed in N2 animals using double-stranded RNA mediated interference we obtained early eggs that showed a strikingly similar phenotype to e2406 embryos at the non-permissive temperature. It is possible that this gene may be a non-muscle isoform that plays an important role in either the specification or regulation of the acto-myosin contractile apparatus involved in cytokinesis. To confirm that this gene corresponds to stu-4 we would like to sequence e2406, and further alleles which we are currently in the process of recovering. In a non-complementation screen singled F1 males from EMS-mutagenized him-8 hermaphrodites were crossed to stu-4(e2406) dyp-5(e61)/szT1 hermaphrodites and we looked for the presence of non dpy sterile progeny. From 740 haploid genomes screened we have obtained one additional allele which we are currently in the process of backcrossing.

Badrinath AS et al. (1996) Midwest Worm Meeting "DYNAMICS OF MITOCHONDRIAL DISTRIBUTION IN EARLY C. ELEGANS EMBRYOS"

White JG, Badrinath AS

[Midwest Worm Meeting, 1996]

Mitochondria are ubiquitous organelles of eukaryotic cells. We would like to understand how they are partitioned to daughter cells during cell division. Specifically, how do the asymmetric cell divisions that take place in the development of C. elegans embryos affect the distribution of mitochondria? Is there any correlation between mitochondrial aggregation and/or activation with a particular cell lineage and/or tissue type? Precedents for both mechanisms have been described. For example, in ascidian embryos the myoplasm, which determines muscle cells (Zalokar M. and Sardet C. (1984) Dev. Biol. 102, 195-205), and the polar lobes in mollusca (Reverberi G. (1970) Acta Embryol. Exp. 1970, 31-43) , which determine mesodermal cells, both contain high densities of mitochondria. Alternatively, in early Drosophila embryos the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of the periplasm, and this changes during development. (Akiyama et al. (1992) Development 115, 1175-1182). Mitochondria are labelled with a mitochondria-specific probe, Rhodamine 6G (R6G), by growing worms on 10 ug/ml R6G-NGM plates. Embryos at pronuclear migration stage are dissected out of the worms and their mitochondria are visualized using a confocal microscope. A single focal plane over time is then animated using NIH image software. Following the first division, P1 appears brighter than AB. Time-lapse movies show that at the time of pseudocleavage there is a flow of mitochondria towards the posteriorly located sperm pronucleus deep in the embryo with a simultaneous cortical flow of mitochondria in the opposite direction. The mitochondria appear to aggregate in the cortical region of the posterior half of the embryo, and this may represent a trapping of some sort. The cortex in the anterior half of the embryo appears to get progressively depleted of mitochondria during the same time period. We are in the process of characterizing this localization of mitochondria in wild-type embryos more carefully, and would like to know if the flow of mitochondria observed is part of the cytoplasmic reorganization that takes place in the first cell cycle (Hird S. and White J.G. (1993) Journal of Cell Biology 121(6) 1343-1355). We would also like to identify mutations that affect mitochondrial distribution. anc-1 (abnormal nuclear ANChorage) is a mutation which affects anchorage of hypodermal nuclei and mitochondria. We are looking at early embryos of anc-1 worms to see if the distribution of mitochondria is affected. This might give us a clue as to how mitochondria appear to get trapped in the posterior cortex. We will also be looking at distributions of mitochondria in worms with par (abnormal cytoplasmic PARtitioning) mutations.

Badrinath AS et al. (1997) International C. elegans Meeting "DYNAMICS OF MITOCHONDRIAL DISTRIBUTION IN EARLY C. ELEGANS EMBRYOS"

White JG, Badrinath AS

[International C. elegans Meeting, 1997]

We have been using fluorescent probes to study the movements of mitochondria during the first cell division. Mitochondria were labelled with a mitochondria-specific probe, Rhodamine 6G (R6G), by growing worms on 2.5 ug/ml R6G-NGM (nematode growth medium) plates. Embryos at pronuclear migration stage were dissected out of the worms and their mitochondria were visualized using a confocal microscope. Image analysis was performed using NIH Image software. Following the first cell division, the mean fluorescence intensity in the posterior blastomere, P1, was found to be approximately 20 percent greater than in the anterior blastomere, AB. Preceding this division, during a stage known as pseudocleavage, mitochondria deep within the embryo flowed posteriorly toward the sperm pronucleus (at a rate of 11.84 +/- 2.37 um/min (n=14, 3 embryos)), and mitochondria located close to the cortex flowed in the opposite direction (at a rate of 14.09 +/- 3.71 um/min (n=19, 3 embryos)). In contrast to these flows that take place in the posterior half of the embryo, there does not appear to be any directed flow of mitochondria in the anterior half either deep within the embryo or close to the cortex. Time-lapse images taken through a cortical section of the embryo clearly show the change in distribution of mitochondria that occurs during pseudocleavage-the flows appear to either enrich the posterior cortex and/or deplete the anterior cortex of mitochondria. We suspect that there might be a mechanism involved in !trapping! mitochondria to the posterior cortex during pseudocleavage, and that this could possibly explain how the difference in mean fluorescence intensity between P1 and AB blastomeres is established. We are currently in the process of obtaining electron micrographs of early embryos in order to confirm the change in distribution of mitochondria observed at the light microscopic level, as well as to reveal any associations between mitochondria and the cytoskeletal elements.

Badrinath AS et al. (2000) Midwest Worm Meeting "Toward the cloning of oj56, a MEL showing variable defects including a failure in cytokinesis."

White JG, Badrinath AS

[Midwest Worm Meeting, 2000]

A small-scale non-complementation screen was performed to isolate new alleles of stu-4. While we did not recover any new alleles as hoped, we were surprised that we had picked up a new non-temperature sensitive maternal effect embryonic lethal mutant, oj56. Homozygous oj56 adults obtained by maternal rescue are easily identified as uncoordinated coilers that are a bit small in size. They are never sterile, but lay approximately 90 percent dead eggs. The survivors either die as larvae, or grow very poorly. The early embryonic defects are quite variable and include delays in cell cycle progression, and what appears to be a failure in the late stages of cytokinesis. Using deficiency mapping we have placed oj56 to the right arm of LG I between gld-1 and mei-1. This is a small region covered by 23 cosmids, and experiments are underway to rescue oj56 by transformation.

Badrinath AS et al. (1998) Worm Breeder's Gazette "Different patterns of mitochondrial redistribution in early C. elegans and Acrobeloides sp. PS1146 embryos."

White JG, Badrinath AS

[Worm Breeder's Gazette, 1998]

The redistribution of mitochondria in the period following fertilization through the first cell division was followed in the early embryos of two related nematodes, Caenorhabditis elegans and Acrobeloides sp. PS1146, using a combination of vital staining with a mitochondrial-specific dye Rhodamine 6G, and confocal laser-scanning microscopy. In C. elegans embryos mitochondria are part of the same bulk flow directed toward the sperm pronucleus that has been implicated in generating the initial A-P axis asymmetry (Strome, S. And Wood, W.B. Cell 35, 15-25 (1983); Hird, S.N. And White, J.G. J. Cell Biology 121, 1343 1355 (1993)). Concomitant with these flows, during the period of cytoplasmic rearrangement known as !pseudocleavage!, most of the cortical mitochondria are found only in the posterior, and following the first cell division the posterior blastomere P1 inherits roughly twenty per cent more mitochondria than its anterior sister AB. This redistribution does not require the presence of either an anterior actin cap or a pseudocleavage since the same pattern was observed in nop-1 mutant embryos in which these structures are missing. In contrast, in Acrobeloides sp. PS1146 embryos the sperm pronucleus does not direct any cytoplasmic rearrangements, and the P-granules are segregated differently. During pronuclear migration they do not become asymmetrically distributed. Most are on the surface of the pronuclei, and the rest are loosely distributed throughout the cytoplasm. During metaphase most of the P-granules are situated around the chromosomes, and by anaphase they redistribute primarily around one of the two microtubule asters (Goldstein, B. et al., Current Biology 8(5): 303. 1998 Feb 26). We found that during pronuclear migration the mitochondria are either around the pronuclei or distributed throughout the cytoplasm, and following the formation of the first spindle they are seen to translocate in radial paths toward the aster centers. This probably indicates transport along astral microtubules mediated by minus-end directed motors. Comparing the two species studied we find that the pattern of redistribution of mitochondria correlates closely with that of the P-granules.

AS Badrinath et al. (1999) International C. elegans Meeting "Cloning and Characterization of the Cytokinesis mutant stu-4."

AS Badrinath, JG White

[International C. elegans Meeting, 1999]

We are characterizing the temperature-sensitive mutant stu-4 (e2406) (originally isolated by David Livingstone). When gravid adults are shifted to the non-permissive temperature 100% of the embryos die due to maternal effect lethality. We examined the cause of this lethality using 4-D time-lapse (Nomarski) microscopy. The polar bodies fail to be extruded resulting in several ooctye 'pronuclei' migrating toward the posteriorly located sperm pronucleus where they generally meet at the center of the embryo. Spindle formation, and reformation of the nuclei following chromosome segregation appears to be normal, but at no time point is there any sign of furrowing at the cortex. This results in a multi-nucleated single celled embryo. When L2 or L3 larval stage e2406 animals are shifted to the non-permissive temperature 100% of them become sterile and exhibit an uncoordinated phenotype to a lesser extent. These defects are presumably due to failures in cell divisions in the post-embryonic lineages giving rise to the gonad and ventral nerve cord respectively. We have mapped stu-4 to - 3.20 on LG I between ace-2 and unc-11 . The deficiency qDf3 uncovered stu-4 whereas qDf4 did not. We previously reported that the cosmid K03E5 was able to rescued stu-4 (WBG 15(3): 26). Currently, we are testing this result more rigorously as well as checking other YACs and cosmids in the region. To confirm that this gene corresponds to stu-4 we would like to sequence e2406, and further alleles which we are currently in the process of recovering. In a non-complementation screen singled F1 males from EMS mutagenized him-8 hermaphrodites were crossed to stu-4 (e2406) dpy 5 (e61)/szT1 hermaphrodites and we looked for the presence of non-dpy sterile progeny. From 740 haploid genomes screened we have obtained one additional allele which we are currently in the process of backcrossing.

Badrinath AS et al. (2003) Dev Biol "Contrasting patterns of mitochondrial redistribution in the early lineages of Caenorhabditis elegans and Acrobeloides sp. PS1146."

White JG, Badrinath AS

[Dev Biol, 2003]

We compared the redistribution of mitochondria in the early embryos of Caenorhabditis elegans (C. elegans) and Acrobeloides sp. PS1146 (Acrobeloides)-two nematode species where the mechanisms for embryonic axis specification are different even though subsequent development is remarkably similar (Goldstein et al., 1998). During the first cell cycle of C. elegans, mitochondria move with the bulk cytoplasmic flows that are directed toward the sperm pronucleus and aggregate at the posterior cortex during the period known as "pseudocleavage." In contrast, in Acrobeloides embryos, where prominent cytoplasmic rearrangements are absent, mitochondria that are initially distributed loosely around the pronuclei and the cytoplasm are relocated around the mitotic spindle prior to cell division. Interestingly, this rearrangement is reiterated only in the germline and not the somatic lineage. In both species, the location of the mitochondria immediately prior to cell division correlates with the known location of the germline determinants, P granules, leading us to speculate that they may be associated.

Ananth Badrinath et al. (2001) International C. elegans Meeting "STU-4, the C. elegans homolog of the S. cerevisiae separin ESP1, is required for meiotic and mitotic chromosome segregation during embryonic and post-embryonic cell divisions."

Ananth Badrinath, John G White

[International C. elegans Meeting, 2001]

How do cells coordinate the processes of chromosome segregation and cell division? To better understand this issue we have been studying the classic temperature-sensitive sterile-uncoordinated mutant, stu-4. In stu-4 (e2406) mutants, cells undergo multiple rounds of normal DNA replication and centrosome duplication, but are aberrant in chromosome segregation and cell division resulting in the formation of polyploid cells. However, there is no apparent delay in the progress of subsequent cell cycles. Furthermore, the defect is not restricted to mitotic cells since meiosis is affected as well. The stu-4 gene encodes a protein with homology to the separins, an emerging class of proteases, which act at the Metaphase to Anaphase transition to cleave the cohesins that hold sister-chromatids together. A second function in Anaphase B spindle movement was also shown recently in S. cerevisiae. We are interested in understanding the function of this protein in a higher metazoan since there are subtle but important differences in the mechanisms of chromosome segregation and cell division between them and yeast.

O'Connell KF et al. (1997) International C. elegans Meeting "NEW MUTATIONS AFFECTING THE GENERATION OF ZYGOTIC POLARITY"

White JG, Badrinath AS, O'Connell KF, Maxwell KN

[International C. elegans Meeting, 1997]

Division of the C. elegans zygote is asymmetric, producing daughter cells that differ in size and developmental potential. To produce distinct daughter cell fates, the zygote must be polarized prior to division such that a different complement of cell fate determinants is inherited by each of the daughters. It is currently thought that the unfertilized egg possesses no inherent polarity and that polarity is established only after fertilization. A number of par genes have been described that are required for partitioning of developmental instructions during the early cleavages and it is thought that the products of these genes interact directly or indirectly with microfilament and microtubule systems to establish polarity and cleavage orientation. Here we describe two new mutations that affect zygotic polarity, both identified in screens for conditional mutations producing a sterile uncoordinated phenotype. oj29 embryos lack functional centrosomes and a number of indicators of embryonic polarity; membrane ruffling and pronuclear migration are aberrant and the posterior-directed cytoplasmic streaming seen in wild-type embryos is missing. stu-4(e2406) isolated by David Livingstone appears to affect the actin cytoskeleton; embryos are defective in polar body formation, pronuclear migration, pseudocleavage and cytokinesis. In addition the first mitotic spindle is often misaligned. We are currently analyzing these mutants for defects in other aspects of asymmetry such as the segregation of P granules. In addition we are screening a small bank of emb mutations, produced in the sterile unc screen, for similar phenotypes.

Wender, Nora et al. (2011) International Worm Meeting "Analysis of cellular toxicity mechanisms of alpha-synuclein in C. elegans."

Wender, Nora, Hegermann, Jan, Eimer, Stefan

[International Worm Meeting, 2011]

Aggregation of a-synuclein (aS) in Lewy bodies is one of the pathological hallmarks of Parkinson's disease (PD). Mutations in aS as well as increased aS expression levels have been associated with PD. As the PD mutants of aS are more prone to forming fibrillar aggregates, these fibrils were initially considered to be causing toxicity. However, in different model systems no clear correlation between toxicity and fibril formation was found. This indicates that small oligomeric precursors rather than fibrils might be the toxic species. In order to investigate which form of aS is causing toxicity, we were making use of a designed variant of aS that stops at the stage of soluble oligomers. This variant is called TP aS (triple proline aS) as it was derived from wt aS by introduction of three proline residues. In vitro NMR studies as well as in vivo analysis of the aggregation of aS variants tagged with YFP in C. elegans muscle cells confirmed that the occurrence of aS fibrils was strongly reduced in TP aS. Next we were assessing toxicity of TP aS in comparison to wt aS as well as PD mutants of aS by investigation of neurite degeneration and impairment of dopamine-associated behavior upon expression of different aS variants in C. elegans dopaminergic neurons. We found that TP is the most toxic aS variant, exhibiting the most severe effects on dopaminergic neuron morphology and function. This suggests that indeed the small oligomers rather than aS fibrils are causing toxicity. So far, the cellular function of aS and the mechanisms causing toxicity remain unclear. It is known that aS binds to intracellular membranes including the mitochondrial membrane. Furthermore, mitochondrial impairment upon expression of wt and mutant aS was reported. Recently it has been reported that several PD-associated genes play a role in mitochondrial dynamics and that loss of function or overexpression leads to morphological changes in the mitochondrial network. Therefore we aimed in understanding how expression of different variants of aS affects mitochondrial dynamics. Thus we were analyzing mitochondrial morphology and distribution by Electron Microscopy as well as Spinning Disk Microscopy. We found that expression of aS in C. elegans muscles and neurons leads to mitochondrial fragmentation and severe changes in mitochondrial morphology. Remarkably, similar changes could be observed in aged worms not expressing aS. It is therefore tempting to suggest that increased expression levels of aS accelerate mitochondrial aging.