Biology subjects

Winkelmeier, J.

Publications and source records attributed to Winkelmeier, J..

3 recordsLinked to original sources

bioRxiv2021

Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color single-molecule localization microscopy

The key to ensuring proper chromosome segregation during mitosis is the kinetochore complex. This large and tightly regulated multi-protein complex links the centromeric chromatin to the microtubules attached to the spindle pole body and as such leads the segregation process. Understanding the architecture, function and regulation of this vital complex is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in high-resolution structural detail so far. In this study we construct a nanometer-precise in situ map of the human-like regional kinetochore of Schizosaccharomyces pombe (S. pombe) using multi-color single-molecule localization microscopy (SMLM). We measure each kinetochore protein of interest (POI) in conjunction with two reference proteins, cnp1CENP-A at the centromere and sad1 at the spindle pole. This arrangement allows us to determine the cell cycle and in particularly the mitotic plane, and to visualize individual centromere regions separately. From these data, we determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI for individual chromosomes and, consequently, build an in situ kinetochore model for S.pombe with so-far unprecedented precision. Being able to quantify the kinetochore proteins within the full in situ kinetochore structure, we provide valuable new insights in the S.pombe kinetochore architecture.

microbiology↗

bioRxiv2020

CAP1 and cofilin1 cooperate in neuronal actin dynamics, growth cone function and neuron connectivity

Neuron connectivity depends on growth cones that navigate axons through the developing brain. Growth cones protrude and retract actin-rich structures to sense guidance cues. These cues control local actin dynamics and steer growth cones towards attractants and away from repellents, thereby directing axon outgrowth. Hence, actin binding proteins (ABPs) moved into the focus as critical regulators of neuron connectivity. We found cyclase-associated protein 1 (CAP1), an ABP with unknown brain function, abundant in growth cones. Super-resolution microscopy and live cell imaging combined with pharmacological approaches on hippocampal neurons from gene-targeted mice revealed a crucial role for CAP1 in actin dynamics that is critical for growth cone morphology and function. Growth cone defects in mutant neurons compromised neuron differentiation and was associated with impaired neuron connectivity in CAP1 mutant brains. Mechanistically, we found that CAP1 and cofilin1 synergistically control growth cone actin dynamic and morphology. Together, we identified CAP1 as a novel actin regulator in growth cone that is relevant for neuron connectivity.

neuroscience↗

bioRxiv2020

Post-mitotic expansion of cell nuclei requires ACTN4-mediated nuclear actin filament bundling

The actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, as well as membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation. Here, we identify non-muscle ACTN4 as a critical regulator to facilitate F-actin formation, reorganization and bundling during postmitotic nuclear expansion. ACTN4 binds to nuclear actin filaments and ACTN4 clusters associate with nuclear F-actin in a highly dynamic fashion. ACTN4 but not ACTN1 is required for proper postmitotic nuclear volume expansion, mediated by its actin binding domain. Using super-resolution imaging to quantify actin filament numbers and widths in individual nuclei we find that ACTN4 is necessary for postmitotic nuclear actin assembly and actin filament bundling. Our findings uncover a nuclear cytoskeletal function for ACTN4 to control nuclear size during mitotic cell division.

cell biology↗