Biomedical subjects

Yvonne Reinders

Publications and source records attributed to Yvonne Reinders.

4 recordsLinked to original sources

PubMed2006

Identification and isolation of Dictyostelium microtubule-associated protein interactors by tandem affinity purification.

Tandem affinity purification (TAP) is a method originally established in yeast to isolate highly purified protein complexes in a very gentle and efficient way. In this work, we have modified TAP for Dictyostelium applications and have proved it as a useful method to specifically isolate and identify microtubule-associated protein (MAP) complexes. MAPs are known to interact with other proteins to fulfill their complex functions in balancing the dynamic instability of microtubules as well as anchoring microtubules at the cell cortex, controlling mitosis at the centrosome and guiding transport along them. DdEB1 and the Dictyostelium member of the XMAP215 protein family, DdCP224, are known to be part of complexes at the microtubule tips as well as at the centrosome. Employing TAP and mass spectrometry we were able to prove an interaction between EB1 and the DdCP224. Additionally, among other interactions that remain to be confirmed by other methods, an interaction between DdCP224 and a TACC-family protein could be shown for the first time in Dictyostelium and was confirmed by colocalization and co-immunoprecipitation analyses.

Animals↗

PubMed2006

Identification of novel centrosomal proteins in Dictyostelium discoideum by comparative proteomic approaches.

The centrosome functions as the main microtubule-organization center of the cell and is of importance for all microtubule-dependent processes such as organelle transport and directionality of cell migration. One of the major model organisms in centrosome research is the slime mold Dictyostelium discoideum. Since only 10 centrosomal proteins are known so far in Dictyostelium discoideum, the elucidation of new centrosomal components may give a more comprehensive understanding of centrosomal function. To distinguish between centrosomal and contaminating proteins we established different separation and relative quantification strategies including techniques such as iTRAQ and DIGE. In this work, we present the identification of several known components as well as more than 70 new candidates--currently subject of further investigations--for the protein inventory of the Dictyostelium centrosome. Among these protein identifications, 44% represent hypothetical proteins of still unknown function associated with the centrosome.

Animals↗

PubMed2006

An improved method for Dictyostelium centrosome isolation.

The Dictyostelium dicoideum centrosome consists of a box-shaped, layered core structure surrounded by dense nodules embedded in amorphous material, which make up the so-called corona. Thus, it differs markedly from centriole-containing centrosomes in animal cells or the plaque structure of yeast spindle pole bodies. For a long time, purification of Dictyostelium centrosomes was hampered by its extraordinarily tight linkage to the nucleus, which resisted all attempts to dissociate centrosomes and nuclei without destruction of the centrosome itself. Fortunately, we were able to solve this problem, and have already published a centrosome isolation protocol that is based on treatment of nucleus/centrosome complexes with sodium pyrophosphate and shear forces, followed by centrosome isolation through sedimentation and filtration techniques. However, isolated centrosomes prepared according to this protocol still contained too many impurities to allow mass spectrometrical analyses. Here, we present an improved protocol for the isolation of Dictyostelium centrosomes that contain considerably less contaminations with cytosolic and nuclear proteins.

Animals↗

PubMed2005

Mass spectrometry-based peptide quantification: applications and limitations.

In various areas of research, proteomics and particularly the quantification of proteins and peptides renders a useful addition to biochemical experiments. The range of possible applications varies from supervision of concentration changes of relevant proteins during biogenesis to differential proteomics approaches, distinguishing, for instance, healthy and diseased states. Furthermore, mass spectrometry-based peptide quantification yields the possibility of using highly sensitive bottom-up approaches for determination of protein regulations as well as multiplexing capability. Thereby, changes in protein abundances may be linked to specific cellular states bearing the opportunity to reveal marker proteins for several diseases.

Amino Acids↗