In this article the calcium/calmodulin-dependent protein kinases are reviewed. The primary focus is on the structure and function of this diverse family of enzymes, and the elegant regulation of their activity. Structures are compared in order to highlight the conserved architecture of their catalytic domains with respect to each other as well as protein kinase A, a prototype for kinase structure. In addition to reviewing structure and function in these enzymes, the variety of biological processes for which they play a mediating role are also examined. Finally, how the enzymes become activated in the intracellular setting is considered by exploring the reciprocal interactions that exist between calcium binding to calmodulin when interacting with the CaM-kinases.

Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFP SW ) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, "patchy" localization patterns of MreB-RFP SW , even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.

The exocytic pathway provides a physical route through which newly synthesized secretory and membrane proteins are deployed to the eukaryote cell surface. For newly synthesized ␣1-antitrypsin (AAT), the modification of its asparagine-linked oligosaccharides by a slow-acting mannosidase partitions the misfolded monomer into the proteasomal degradation pathway. Herein, we asked whether, and how, modification by endoplasmic reticulum mannosidase I (ERManI) contributes to the preferential selection of the misfolded AAT monomer for proteasomal degradation. Transiently expressed mutant and WT AAT variants underwent rapid destabilization in response to an artificially elevated ERManI concentration in the murine hepatoma cell line, Hepa1a. Based on the mannosidase-and lactacystin-sensitive properties of intracellular turnover, a stochastic model is proposed in which the delayed onset of the glycan modification, relative to the duration of nonnative protein structure, coordinates the preferential degradation of the misfolded monomer and spares the native molecule from destruction. Newly synthesized endogenous transferrin underwent degradation in response to an elevated concentration of ERManI, whereas the nonglycosylated secretory glycoprotein albumin was not affected. Taken together, these findings indicate that efficient conformational maturation might function as the initial quality control standard for a broad population of glycoproteins.

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (>80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.

Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of ∼150 nm in diameter that "saddles" the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane, and angular distribution with respect to the membrane.C ytokinesis, the final step of cell division in eukaryotic cells, is typically driven by a contractile actomyosin ring (AMR) primarily composed of actin (1) and myosin (2). Our understanding of the molecular mechanisms of cytokinesis is most detailed in the rodshaped unicellular eukaryote Schizosaccharomyces pombe (otherwise known as fission yeast), which shares a remarkably conserved set of cytokinesis genes with metazoans (3). In S. pombe, the AMR undergoes multiple phases known as assembly, maturation, constriction, and disassembly (4), with open questions in each of these four stages. Due to a lack of information about the precise arrangement of filamentous actin (F-actin) within the force-generating network of the AMR, we chose to focus on imaging the AMR during constriction.In S. pombe, glancing sections through plastic-embedded, dividing cells gave the first glimpse of actin filaments running parallel to the division plane at the front of the septum (5). Unfortunately, the study yielded limited examples and lacked 3D information for a full analysis. In an ambitious pioneering effort, Kamasaki et al. (6) produced 3D reconstructions of entire S. pombe AMRs by imaging serial sections through permeabilized cells decorated with myosin S1 fragments. The amount of F-actin and the size of the rings appeared significantly altered by the procedure used for preserving them (details in Discussion), but the continuous bundles that were reconstructed were composed of mixed polarity filaments running circumferentially around the cell.Here we sought to visualize the precise arrangement of F-actin within the AMR and its interface with the membrane by imaging intact cells in a cryopreserved state using electron cryotomography (ECT) (7). Because whole S. pombe cells are too thick for ECT, which is limited to specimens thinner than a few hundred nanometers, we overcame this obstacle by first rapid freezing dividing cells and then either cryosectioning them or using the recently developed method cryofocused ion beam (cryo-FIB) milling to ...