During animal cell division, the cleavage furrow is positioned by microtubules that signal to the actin cortex at the cell midplane. We developed a cell-free system to recapitulate cytokinesis signaling using cytoplasmic extract from Xenopus eggs. Microtubules grew out as asters from artificial centrosomes and met to organize antiparallel overlap zones. These zones blocked interpenetration of neighboring asters and recruited cytokinesis midzone proteins including the Chromosomal Passenger Complex (CPC) and Centralspindlin. The CPC was transported to overlap zones, which required two motor proteins, Kif4A and a Kif20A paralog. Using supported lipid bilayers to mimic the plasma membrane, we observed recruitment of cleavage furrow markers, including a RhoA-GTP reporter, at microtubule overlaps. This system opens further approaches to understanding the biophysics of cytokinesis signaling.

The M2 protein from influenza A is a pH-activated proton channel that plays an essential role in the viral life cycle and serves as a drug target. Using spin labeling EPR spectroscopy we studied a 38-residue M2 peptide spanning the transmembrane region and its C-terminal extension. We obtained residue-specific environmental parameters under both high and low pH conditions for nine consecutive C-terminal sites. The region forms a membrane surface helix at both high and low pH although the arrangement of the monomers within the tetramer changes with pH. Both electrophysiology and EPR data point to a critical role for residue Lys 49.M2 is a 96-residue homotetrameric integral membrane protein with a small N-terminal ectodomain, a single transmembrane helix and a C-terminal cytoplasmic tail. Despite data from solid state NMR (1), x-ray crystallography (2) and solution NMR (3), a detailed understanding of how the M2 protein works continues to puzzle investigators and generate sharp controversy.The majority of published studies on the proton channel function of M2 have focused on the transmembrane (TM) 1 domain. However, truncation studies indicate that the cytoplasmic domain also plays a role in channel stability (4). Proteolysis of micelle-bound full length M2 revealed that a 15-20 residue segment C-terminal to the TM helix was highly protected from cleavage by proteases (5). Helical wheel analysis of the protected region (5) suggested that the segment could form an amphiphilic helix, consistent with later findings from solid state NMR on M2 protein in lipid bilayers (6). In order to further test the proposed models, we probed the conformation of the segment C-terminal to the TM domain at both high and low pH using sitedirected spin-labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy.EPR studies were performed on a series of 38-residue synthetic M2 peptides (residues 23-60; M2TMC) spanning the TM region and the beginning of the C-terminal domain. We spin- † This research was supported by R01AI57363 (LHP), GM56423 (WFD), a Henry Dreyfus Teacher Scholar Award (KPH) and R15AI074033 (KPH).

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