Empowering statistical methods for cellular and molecular biologists.

We provide guidelines for using statistical methods to analyze the types of experiments reported in cellular and molecular biology journals such as Molecular Biology of the Cell. Our aim is to help experimentalists use these methods skillfully, avoid mistakes, and extract the maximum amount of information from their laboratory work. We focus on comparing the average values of control and experimental samples. A Supplemental Tutorial provides examples of how to analyze experimental data using R software

The Obligation for Biologists to Commit to Political Advocacy

I explain here why all scientists should feel obligated to do their part to support the community by advocating for the benefits of government investments in scientific research and training

Distinct Roles for F-BAR Proteins Cdc15p and Bzz1p in Actin Polymerization at Sites of Endocytosis in Fission Yeast

SummaryBackgroundGenetic analyses of budding and fission yeast identified >50 proteins that assemble at sites of clathrin-mediated endocytosis in structures called actin patches. These proteins include clathrin, clathrin-interacting proteins, actin binding proteins, and peripheral membrane proteins such as F-BAR proteins. Many questions remain regarding the interactions of these proteins, particularly the participation of F-BAR proteins in the assembly of actin filaments.ResultsOur microscopic and genetic interaction experiments on fission yeast show that F-BAR proteins Cdc15p and Bzz1p accumulate in two distinct zones on invaginating membrane tubules and interact with Myo1p and Wsp1p, nucleation-promoting factors for Arp2/3 complex. The two F-BAR proteins peak prior to movement of the actin patch and their accumulation in actin patches depends on the nucleation-promoting factors. At their peak local concentrations, we estimated the stoichiometries of the proteins in actin patches to be one Bzz1p per two Wsp1p and one Cdc15p per Myo1p. Purified Bzz1p has two SH3 domains that interact with Wsp1p and stimulate actin polymerization by Arp2/3 complex. Cells lacking either Cdc15p or Bzz1p assemble 3- to 5-fold less actin in patches (in spite of normal levels of Wsp1p, Myo1p, and Arp2/3 complex), and patches move shorter distances from the plasma membrane.ConclusionWe propose that during clathrin-mediated endocytosis, F-BAR proteins interact with nucleation-promoting factors to stimulate Arp2/3 complex in two different zones along the invaginating tubule. We further propose that polymerization of actin filaments in these two zones contributes to membrane scission

Kinetics of the Formation and Dissociation of Actin Filament Branches Mediated by Arp2/3 Complex

AbstractThe actin filament network at the leading edge of motile cells relies on localized branching by Arp2/3 complex from “mother” filaments growing near the plasma membrane. The nucleotide bound to the mother filaments (ATP, ADP and phosphate, or ADP) may influence the branch dynamics. To determine the effect of the nucleotide bound to the subunits of the mother filament on the formation and stability of branches, we compared the time courses of actin polymerization in bulk samples measured using the fluorescence of pyrene actin with observations of single filaments by total internal reflection fluorescence microscopy. Although the branch nucleation rate in bulk samples was nearly the same regardless of the nucleotide on the mother filaments, we observed fewer branches by microscopy on ADP-bound filaments than on ADP-Pi-bound filaments. Observation of branches in the microscope depends on their binding to the slide. Since the probability that a branch binds to the slide is directly related to its lifetime, we used counts of branches to infer their rates of dissociation from mother filaments. We conclude that the nucleotide on the mother filament does not affect the initial branching event but that branches are an order of magnitude more stable on the sides of new ATP- or ADP-Pi filaments than on ADP-actin filaments

UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis