Biology subjects

Rusan, N. M.

Publications and source records attributed to Rusan, N. M..

4 recordsLinked to original sources

bioRxiv2018

Fascetto Interacting Protein (FIP) Regulates Fascetto (PRC1) to Ensure Proper Cytokinesis and Ploidy

Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring, and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is Protein Regulator of Cytokinesis 1 (PRC1), or Fascetto (Feo) in Drosophila, which forms a homodimer to crosslink interzonal MTs, ensuring proper central spindle formation and cytokinesis. Here, we report on a new direct protein interactor and regulator of Feo we named Fascetto Interacting Protein (FIP). Loss of FIP results in a significant reduction in Feo localization, rapid disassembly of interzonal MTs, and several cytokinesis defects. Simultaneous reduction in Feo and FIP results in tumor-like, DNA-filled masses in the brain. In aggregate our data show that FIP functions upstream of, and acts directly on, Feo to ensure fully accurate cell division.

cell biology↗

bioRxiv2018

Micro computed tomography as an accessible imaging platform for exploring organism development and human disease modeling

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here we present an optimized method for non-invasive imaging of Drosophila melanogaster at high resolution using micro computed tomography (-CT). Our method allows for rapid processing of intact animals at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use the power of -CT imaging to model human diseases through the characterization of microcephaly in the fly. Our work demonstrates that -CT is a versatile and accessible tool that complements standard imaging techniques, capable of uncovering novel biological mechanisms that have remained undocumented due to limitations of current methods.

developmental biology↗

bioRxiv2017

An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event, involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 binding stimulates Plk4 kinase activity in vitro, and, in turn, is phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STAN domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N-terminus of Ana2 which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.

cell biology↗

bioRxiv2017

The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes a significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution interactions are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results we suggest a model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix.

biochemistry↗