Valerie C. Coffman

Assembly and architecture of precursor nodes during fission yeast cytokinesis

Mapping of fission yeast precursor node interaction modules and assembly reveals important steps in contractile ring assembly.

CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast.

Current models of centromere/kinetochore architecture are not sufficient to explain the number of molecules of histone H3 variant CENP-A observed in quantitative microscopy.

Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30-50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. alpha-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis.

Counting protein molecules using quantitative fluorescence microscopy

In recent years, quantification of absolute protein numbers in cellular structures using fluorescence microscopy has become a reality. Two popular methods are available to a broad range of researchers with minimal equipment and analysis requirements: stepwise photobleaching to count discrete changes in intensity from a small number of fluorescent fusion proteins, and comparing the fluorescence intensity of a protein to a known in vivo or in vitro standard. This review summarizes the advantages and disadvantages of each method, and gives recent examples of each that answer important questions in their respective fields. We also highlight new counting methods that could become widely available in the future.

Contractile‐ring assembly in fission yeast cytokinesis: Recent advances and new perspectives

The fission yeast Schizosaccharomyces pombe is an excellent model organism to study cytokinesis. Here, we review recent advances on contractile‐ring assembly in fission yeast. First, we summarize the assembly of cytokinesis nodes, the precursors of a normal contractile ring. IQGAP Rng2 and myosin essential light chain Cdc4 are recruited by the anillin‐like protein Mid1, followed by the addition of other cytokinesis node proteins. Mid1 localization on the plasma membrane is stabilized by interphase node proteins. Second, we discuss proteins and processes that contribute to the search, capture, pull, and release mechanism of contractile‐ring assembly. Actin filaments nucleated by formin Cdc12, the motor activity of myosin‐II, the stiffness of the actin network, and severing of actin filaments by cofilin all play essential roles in contractile‐ring assembly. Finally, we discuss the Mid1‐independent pathway for ring assembly, and the possible mechanisms underlying the ring maturation and constriction. Collectively, we provide an overview of the current understanding of contractile‐ring assembly and uncover future directions in studying cytokinesis in fission yeast.

The formins Cdc12 and For3 cooperate during contractile ring assembly in cytokinesis.

Multiple formins cooperate during cytokinesis, but their functions in de novo actin assembly at the division site play the primary role in contractile ring assembly.

Every laboratory with a fluorescence microscope should consider counting molecules

Protein numbers in cells determine rates of biological processes, influence the architecture of cellular structures, reveal the stoichiometries of protein complexes, guide in vitro biochemical reconstitutions, and provide parameter values for mathematical modeling. The purpose of this essay is to increase awareness of methods for counting protein molecules using fluorescence microscopy and encourage more cell biologists to report these numbers. We address the state of the field in terms of utility and accuracy of the numbers reported and point readers to references for details of specific techniques and applications.

Stronger net posterior cortical forces and asymmetric microtubule arrays produce simultaneous centration and rotation of the pronuclear complex in the early Caenorhabditis elegans embryo

Experimental and theoretical approaches are used to demonstrate the importance of asymmetries in microtubule arrays and cortical pulling forces mediated by dynein in positioning the pronuclear complex before nuclear envelope breakdown in the early Caenorhabditis elegans embryo.

Antagonistic Behaviors of NMY-1 and NMY-2 Maintain Ring Channels in the C. elegans Gonad

Contractile rings play critical roles in a number of biological processes, including oogenesis, wound healing, and cytokinesis. In many cases, the activity of motor proteins such as nonmuscle myosins is required for appropriate constriction of these contractile rings. In the gonad of the nematode worm Caenorhabditis elegans, ring channels are a specialized form of contractile ring that are maintained at a constant diameter before oogenesis. We propose a model of ring channel maintenance that explicitly incorporates force generation by motor proteins that can act normally or tangentially to the ring channel opening. We find that both modes of force generation are needed to maintain the ring channels. We demonstrate experimentally that the type II myosins NMY-1 and NMY-2 antagonize each other in the ring channels by producing force in perpendicular directions: the experimental depletion of NMY-1/theoretical decrease in orthogonal force allows premature ring constriction and cellularization, whereas the experimental depletion of NMY-2/theoretical decrease in tangential force opens the ring channels and prevents cellularization. Together, our experimental and theoretical results show that both forces, mediated by NMY-1 and NMY-2, are crucial for maintaining the appropriate ring channel diameter and dynamics throughout the gonad.