We used fluorescence microscopy to measure global and local concentrations of 28 cytoskeletal and signaling proteins fused to yellow fluorescent protein (YFP) in the fission yeast Schizosaccharomyces pombe. Native promoters controlled the expression of these functional YFP fusion proteins. Fluorescence measured by microscopy or flow cytometry was directly proportional to protein concentration measured by quantitative immunoblotting. Global cytoplasmic concentrations ranged from 0.04 (formin Cdc12p) to 63 micromolar (actin). Proteins concentrated up to 100 times in contractile rings and 7500 times in spindle pole bodies at certain times in the cell cycle. This approach can be used to measure the global and local concentrations of any fusion protein.

Microscopy of fluorescent fusion proteins and genetic dependencies show that fission yeast assemble and constrict a cytokinetic contractile ring in a precisely timed, sequential order. More than 90 min prior to separation of the spindle pole bodies (SPB), the anillin-like protein (Mid1p) migrates from the nucleus and specifies a broad band of cortex around the equator as the division site. Between 10 min before and 2 min after SPB separation, conventional myosin-II (Myo2p), IQGAP (Rng2p), PCH protein (Cdc15p), and formin (Cdc12p) join the broad band independent of actin filaments. Over the subsequent 10 min prior to anaphase B, this broad band of proteins condenses into a contractile ring including actin, tropomyosin (Cdc8p), and alpha-actinin (Ain1p). During anaphase B, unconventional myosin-II (Myp2p) joins the ring followed by the septin (Spn1p). Ring contraction and disassembly begin 37 min after SPB separation. This spatial and temporal hierarchy provides the framework for analysis of molecular mechanisms.

Animals and fungi assemble a contractile ring of actin filaments and the motor protein myosin to separate into individual daughter cells during cytokinesis. We used fluorescence microscopy of live fission yeast cells to observe that membrane-bound nodes containing myosin were broadly distributed around the cell equator and assembled into a contractile ring through stochastic motions, after a meshwork of dynamic actin filaments appeared. Analysis of node motions and numerical simulations supported a mechanism whereby transient connections are established when myosins in one node capture and exert force on actin filaments growing from other nodes.