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Kristen M Johansen

Publications and source records attributed to Kristen M Johansen.

8 recordsLinked to original sources
PubMed2004

Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor.

We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.

Amino Acid Sequence↗
PubMed2004

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila.

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.

Animals↗
PubMed2004

Leech filamin and Tractin: markers for muscle development and nerve formation.

The Lan3-14 and Laz10-1 monoclonal antibodies recognize a 400 kDa antigen that is specifically expressed by all muscle cells in leech. We show that the antigen recognized by both antibodies is a member of the filamin family of actin binding proteins. Leech filamin has two calponin homology domains and 35 filamin/ABP-repeat domains. In addition, we used the Laz10-1 antibody to characterize the development of the segmentally iterated dorsoventral flattener muscles. We demonstrate that the dorsoventral flattener muscle develops as three discrete bundles of myofibers and that CNS axons pioneering the DP nerve extend only along the middle bundle. Interestingly, the middle dorsoventral muscle anlage is associated with only non-neuronal expression of the L1-family cell adhesion molecule Tractin. This expression is transient and occurs at the precise developmental stages when DP nerve formation takes place. Based on these findings we propose that the middle dorsoventral muscle anlagen provides a substrate for early axonal outgrowth and nerve formation and that this function may be associated with differential expression of distinct cell adhesion molecules.

Animals↗
PubMed2003

A developmentally regulated splice variant from the complex lola locus encoding multiple different zinc finger domain proteins interacts with the chromosomal kinase JIL-1.

Using a yeast two-hybrid screen we have identified a novel isoform of the lola locus, Lola zf5, that interacts with the chromosomal kinase JIL-1. We characterized the lola locus and provide evidence that it is a complex locus from which at least 17 different splice variants are likely to be generated. Fifteen of these each have a different zinc finger domain, whereas two are without. This potential for expression of multiple gene products suggests that they serve diverse functional roles in different developmental contexts. By Northern and Western blot analyses we demonstrate that the expression of Lola zf5 is developmentally regulated and that it is restricted to early embryogenesis. Immunocytochemical labeling with a Lola zf5-specific antibody of Drosophila embryos indicates that Lola zf5 is localized to nuclei. Furthermore, by creating double-mutant flies we show that a reduction of Lola protein levels resulting from mutations in the lola locus acts as a dominant modifier of a hypomorphic JIL-1 allele leading to an increase in embryonic viability. Thus, genetic interaction assays provide direct evidence that gene products from the lola locus function within the same pathway as the chromosomal kinase JIL-1.

Amino Acid Sequence↗
PubMed2003

Genetic and phenotypic analysis of alleles of the Drosophila chromosomal JIL-1 kinase reveals a functional requirement at multiple developmental stages.

In this study we provide a cytological and genetic characterization of the JIL-1 locus in Drosophila. JIL-1 is an essential chromosomal tandem kinase and in JIL-1 null animals chromatin structure is severely perturbed. Using a range of JIL-1 hypomorphic mutations, we show that they form an allelic series. JIL-1 has a strong maternal effect and JIL-1 activity is required at all stages of development, including embryonic, larval, and pupal stages. Furthermore, we identified a new allele of JIL-1, JIL-1(h9), that encodes a truncated protein missing COOH-terminal sequences. Remarkably, the truncated JIL-1 protein can partially restore viability without rescuing the defects in polytene chromosome organization. This suggests that sequences within this region of JIL-1 play an important role in establishing and/or maintaining normal chromatin structure. By analyzing the effects of JIL-1 mutations we provide evidence that JIL-1 function is necessary for the normal progression of several developmental processes at different developmental stages such as oogenesis and segment specification. We propose that JIL-1 may exert such effects by a general regulation of chromatin structure affecting gene expression.

Alleles↗
PubMed2002

Proteolytic cleavage of the ectodomain of the L1 CAM family member Tractin.

Tractin is a member of the L1 family of cell adhesion molecules in leech. Immunoblot analysis suggests that Tractin is constitutively cleaved in vivo at a proteolytic site with the sequence RKRRSR. This sequence conforms to the consensus sequence for cleavage by members of the furin family of convertases, and this proteolytic site is shared by a majority of other L1 family members. We provide evidence with furin-specific inhibitor experiments, by site-specific mutagenesis of Tractin constructs expressed in S2 cells, as well as by Tractin expression in furin-deficient LoVo cells that a furin convertase is the likely protease mediating this processing. Cross-immunoprecipitations with Tractin domain-specific antibodies suggest that the resulting NH(2)- and COOH-terminal cleavage fragments interact with each other and that this interaction provides a means for the NH(2)-terminal fragment to be tethered to the membrane. Furthermore, in S2 cell aggregation assays we show that the NH(2)-terminal fragment is necessary for homophilic adhesion and that cells expressing only the transmembrane COOH-terminal fragment are non-adhesive. However, tethering of exogeneously provided Tractin NH(2)-terminal fragment to S2 cells expressing only the COOH-terminal fragment can functionally restore the adhesive properties of Tractin.

Animals↗
PubMedDate not supplied

Recent glimpses of the elusive spindle matrix.

A stationary spindle matrix has been proposed on theoretical grounds to help mediate force production at the mitotic spindle. Direct molecular evidence for the existence of such a matrix has the potential to profoundly influence our view of the molecular mechanisms leading to chromosome segregation during mitosis. Three recent papers suggest that the spindle matrix may be more than a theoretical idea.

Animals↗