Higher-ordered assembly of septins in a cell-based system.

点击翻译按钮获取中文摘要

Septins form a family of highly conserved, cytoskeletal proteins that act at the interface of membranes and the actin- and microtubule-based cytoskeletons. Known to hetero and homo oligomerize into filaments and higher-ordered structures such as bundled filaments and rings, septins scaffold other proteins and can act as membrane diffusion barriers in diverse cellular structures such as the base of the primary cilium, the sperm annulus, and the cleavage apparatus during cell division. The human genome encodes fourteen septins, while Drosophila has just five septin genes: sep1, sep2, sep4, sep5 and peanut. We have discovered that Drosophila Schneider's S2 cells form cytoplasmic, higher-ordered tubular structures up to several microns in length that label for Peanut and Sep2, the only septins thus far tested. These cytoplasmic tubes do not appear to be cell-cycle regulated and do not co-localize with F-actin or microtubules. However they disassemble into rings when F-actin polymerization is inhibited, indicating indirect, but functional interactions with the actin cytoskeleton.**Hypothesis, Aims and Approach*Our long-term aim is to determine how septins influence cellular architecture. We hypothesize that higher-ordered cytoplasmic septin assemblies provide a unique opportunity for understanding septin organization and function in cells. Our short-term aim is thus to develop and exploit this novel cell-based assay.**We will use a Drosophila S2 cell model, with which we have extensive experience, and that combines genetic (RNAi, expression of fluorescent proteins) and pharmacological tools coupled with live-cell fluorescence microscopy, quantitative image analysis and electron microscopy. We will pursue the following specific aims:**1) Define the composition, principles of assembly and dynamic properties of septin tubes and rings. *2) Test potential mechanisms of interaction between the actin and septin cytoskeletons. *3) Identify other regulators of septin tube assembly/disassembly.**These studies will elucidate the molecular mechanisms by which septins can assemble into higher-ordered structures in living cells, and will shed light on the undoubtedly complex interplay between septins and actin in interphasic cells. This will further our basic understanding of the architecture of the metazoan cell.