Biomedical subjects

Carsten Janke

Publications and source records attributed to Carsten Janke.

8 recordsLinked to original sources

PubMed2026

Microtubule posttranslational modifications provide unique recognition patterns for associated proteins.

Microtubules are key components of the eukaryotic cytoskeleton involved in vital functions in virtually every cell. Among the emerging molecular mechanisms to adapt microtubules to their diverse functions is the biochemical diversification of tubulin molecules by posttranslational modifications (PTMs) and differential gene expression, a concept known as the 'tubulin code'. A key question remains whether the tubulin code has the potential to selectively control microtubule interactions of different microtubule-associated proteins (MAPs) to act as a specific signalling system. To answer this question, we used a medium-throughput in vitro approach to screen 46 proteins for their binding preferences to microtubules with altered PTM or isotype composition. We demonstrate that subsets of these MAPs have unique sensitivities to PTMs, while other proteins are not affected. As a result, PTMs, or combinations of them, differentially attract or repulse individual MAPs to microtubules. Our findings offer mechanistic proof for a key hypothesis of the tubulin code-the capacity to selectively and differentially regulate MAP-microtubule interactions.

Journal Article↗

PubMed2005

Tubulin polyglutamylase enzymes are members of the TTL domain protein family.

Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin tyrosine ligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization.

Amino Acid Motifs↗

PubMed2004

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.

Gene Targeting↗

PubMed2003

Nsl1p is essential for the establishment of bipolarity and the localization of the Dam-Duo complex.

We identified a physical complex consisting of Mtw1p, an established kinetochore protein, with Nnf1p, Nsl1p and Dsn1p and have demonstrated that Nnf1p, Nsl1p and Dsn1p localize to the Saccharomyces cerevisiae kinetochore. When challenged prior to metaphase, the temperature-sensitive mutants nsl1-16 and nsl1-42 as well as Nsl1p-depleted cells failed to establish a bipolar spindle-kinetochore interaction and executed monopolar segregation of sister chromatids. In contrast, an nsl1-16 defect could not be evoked after the establishment of bipolarity. The observed phenotype is characteristic of that of mutants with defects in the protein kinase Ipl1p or components of the Dam-Duo kinetochore complex. However nsl1 mutants did not exhibit a defect in microtubule-kinetochore untethering as the ipl1-321 mutant does. Instead, they exhibited a severe defect in the kinetochore localization of the Dam-Duo complex suggesting this to be the cause for the failure of nsl1 cells to establish bipolarity. Moreover the analysis of Nsl1p-depleted cells indicated that Nsl1p is required for the spindle checkpoint and kinetochore integrity.

Cell Cycle Proteins↗

PubMed2003

Characterisation of PGs1, a subunit of a protein complex co-purifying with tubulin polyglutamylase.

Polyglutamylation is a post-translational modification initially discovered on tubulin. It has been implicated in multiple microtubule functions, including neuronal differentiation, axonemal beating and stability of the centrioles, and shown to modulate the interaction between tubulin and microtubule associated proteins. The enzymes catalysing this modification are not yet known. Starting with a partially purified fraction of mouse brain tubulin polyglutamylase, monoclonal antibodies were raised and used to further purify the enzyme by immunoprecipitation. The purified enzyme complex (Mr 360x103) displayed at least three major polypeptides of 32, 50 and 80x103, present in stochiometric amounts. We show that the 32x103 subunit is encoded by the mouse gene GTRGEO22, the mutation of which has recently been implicated in multiple defects in mice, including male sterility. We demonstrate that this subunit, called PGs1, has no catalytic activity on its own, but is implicated in the localisation of the enzyme at major sites of polyglutamylation, i.e. neurones, axonemes and centrioles.

Animals↗

PubMed2003

Drosophila DSP1 and rat HMGB1 have equivalent DNA binding properties and share a similar secondary fold.

The protein DSP1 belongs to the group of HMG-box proteins, which share the common structural feature of the HMG-box. This approximately 80 amino acid long motif binds DNA via the minor groove. DSP1 was discovered as a transcriptional co-repressor of Dorsal in Drosophila melanogaster and then was shown to participate to the remodeling of chromatin. By means of sequence alignment and gene organization, DSP1 was classified as the fly homologue of the vertebrate proteins HMGB1/2. DSP1 contains two HMG boxes flanked by two glutamine-rich domains at the N-terminus. In addition, the HMG domain of DSP1 displays two differences in its primary sequence as compared to the vertebrate HMGB1: a shorter acidic tail and a linker between the two boxes longer by 6 amino acids. By comparing several functional parameters of DSP1 with those of HMGB1, the present study establishes the functional equivalence of both proteins in terms of DNA recognition. The major structural difference between the two proteins, the glutamine-rich N-terminal tail of DSP1, which does not exist in HMGB1, did not interfere with any of the studied DNA-binding properties of the proteins.

Animals↗

PubMed2002

Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections.

How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole. We investigated IPL1 function in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin.

Aurora Kinases↗

PubMed2002

Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister kinetochore biorientation.

We show here that Ask1p, Dad2p, Spc19p and Spc34p are subunits of the budding yeast Duo1p-Dam1p- Dad1p complex, which associate with kinetochores and localize along metaphase and anaphase spindles. Analysis of spc34-3 cells revealed three novel functions of the Duo1-Dam1p-Dad1p subunit Spc34p. First, SPC34 is required to establish biorientation of sister kinetochores. Secondly, SPC34 is essential to maintain biorientation. Thirdly, SPC34 is necessary to maintain an anaphase spindle independently of chromosome segregation. Moreover, we show that in spc34-3 cells, sister centromeres preferentially associate with the pre-existing, old spindle pole body (SPB). A similar preferential attachment of sister centromeres to the old SPB occurs in cells depleted of the cohesin Scc1p, a protein with a known role in facilitating biorientation. Thus, the two SPBs are not equally active in early S phase. We suggest that not only in spc34-3 and Deltascc1 cells but also in wild-type cells, sister centromeres bind after replication preferentially to microtubules organized by the old SPB. Monopolar attached sister centromeres are resolved to bipolar attachment in wild-type cells but persist in spc34-3 cells.

Anaphase↗