Ca2+ signalling (p. 2213 and p. 2223) Signal Transduction and Cellular Organization

The mechanisms by which Ca2+ is released from intracellular stores and enters across the plasma membrane (PM) are important features of the cell biology of this intracellular messenger. They allow cells to generate a variety of distinct spatiotemporal Ca2+ signals and provide a mechanism for maintaining intracellular Ca2+ homeostasis. In this issue, Commentaries in our Signal Transduction and Cellular Organization series focus on two important aspects of Ca2+ release and entry: the generation of ‘local’ Ca2+ signals and capacitative Ca2+ entry.

On p. 2213, Martin Bootman and co-workers review our understanding of the roles of local Ca2+ signals - spatially restricted rises in cytosolic [Ca2+] such as the Ca2+ ‘puffs’ and ‘spikes’ of non-excitable cells and the Ca2+ ‘sparks’ of excitable cells. These local signals vary from 10 nm to several micrometres in diameter and can produce specificity in the response by restricting Ca2+ to particular targets. Adenylyl cyclase, for example, is more sensitive to Ca2+ entering through store-operated Ca2+ channels (SOCCs) than to that entering through voltage-operated Ca2+ channels (VOCCs) - presumably because SOCCs are localized close to the enzyme. Local Ca2+ signals can also trigger much larger signals that spread throughout the cell. For example, coordinated recruitment of Ca2+ puffs arising at sites containing multiple inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptors generates the characteristic Ca2+ waves observed during hormonal stimulation. Local signals can thus not only have local effects in particular microdomains but also have far-reaching effects with long-term consequences.

On p. 2223, Jim Putney and co-workers continue the discussion of Ca2+ signalling, focusing on the mechanism(s) by which depletion of ER Ca2+ stores induces influx of Ca2+ through PM channels - a phenomenon known as capacitative Ca2+ entry. One possibility is that the ER stores regulate PM Ca2+ channels by controlling the level of Ca2+ (which inhibits the channels) in their immediate vicinity. Alternatively, stores might release a diffusible messenger that opens PM Ca2+ channels - indeed, such an activity (calcium-influx factor; CIF) has been isolated. Perhaps the most compelling model, however, is ‘conformational coupling’, in which ER Ins(1,4,5)P3 receptors communicate the filling state of the ER by interacting directly with PM Ca2+ channels. Recent work, including the observation that in excised PM patches certain channels require Ins(1,4,5)P3 receptors for activity, provides strong support for conformational coupling. Nevertheless, since capacitative Ca2+ entry can operate in cells lacking Ins(1,4,5)P3 receptors, Putney and co-workers conclude that multiple mechanisms underlying this phenomenon probably exist.

A scaffold for β1,4-galactosyl-transferase (p. 2291)

β1,4-galactosyltransferase I (GalT) is a transmembrane glycoprotein responsible for galactosylation of certain glycoconjugates in the Golgi. Unlike many glycosyltransferases, GalT is also present at the surface: it acts as a lectin-like matrix receptor that facilitates cell spreading by associating with the cytoskeleton and activating cell-specific signalling cascades. Barry Shur and co-workers have used a two-hybrid approach to identify signalling proteins with which GalT interacts. They show that the cytoplasmic domain of GalT binds to SSeCKS - a PKC target that is homologous to gravin and functions as a scaffolding protein and an organizer of the cytoskeleton. Furthermore, they demonstrate that this unprecedented glycosyltransferase-scaffold interaction occurs in vivo, SSeCKS and GalT colocalizing in both the Golgi and filopodia. The authors show that the interaction is functional, since SSeCKS constructs containing the GalT-interaction sites can reverse the spreading defect caused by a dominant negative GalT mutant. They propose that the SSeCKS scaffold orchestrates the signalling/cytoskeletal functions of GalT, suggesting that its binding could be coupled to GalT-dependent FAK activation and/or recruitment of signalling molecules to the GalT microenvironment.

3D organization of the yeast secretory pathway (p. 2231)

The classic textbook diagram of the Golgi apparatus shows a series of independent flattened sacs. This might be an accurate representation of the mammalian Golgi. But what of the more elusive yeast Golgi? Alain Rambourg, Catherine Jackson and Yves Clermont have studied the Golgi apparatus and other membrane structures of the budding yeast secretory pathway under 3D stereo electron microscopy - employing brefeldin A treatment and sec21 ts mutants to identify the ER and Golgi compartments. They observe that the structures involved are polygonal tubular networks connected to fenestrated ER sheets. Interestingly, the Golgi elements generally appear to exist not as flattened sacs or cisternae but instead as tubular networks - a morphology that segments of the ER can also occasionally adopt. The authors demonstrate that secretory granules are progressively segregated at the intersections of the tubules making up the polygonal networks. The granules are liberated by rupture of these tubular networks rather than, as previously predicted, by budding from the edges of saccular elements. The authors’ findings suggest that cargo transport along the yeast secretory pathway involves vectorial membrane flow through a series of membrane transformations rather than transport between fixed structural compartments.

Control of mitotic exit by Bub2p-Bfa1p (p. 2345)

The spindle assembly checkpoint (SAC) ensures that cells possessing damaged spindles or unattached kinetochores arrest in metaphase and do not exit mitosis. In budding yeast, Bub2p and Bfa1p appear to be key components of the arm of the pathway that blocks mitotic exit. The two proteins might function as a GTPase-activating protein (GAP) that regulates Tem1p - a GTPase that is part of the mitotic exit network (MEN) and is activated by the exchange factor Lte1p. Leland Johnston and co-workers show that Bub2p andBfa1p are present as a complex throughout the cell cycle and associate with Tem1p during M phase and early G1 phase. Furthermore, they show that Lte1p and Bfa1p (but not Bub2p) are phosphorylated in a cell-cycle-dependent manner and after SAC activation - phosphorylation of both proteins being in part due to the Polo-related kinase Cdc5p. The authors propose that, following SAC activation, phosphorylation of Bfa1p by Cdc5p stimulates Bub2p GAP activity, which, combined with inhibitory phosphorylation of Lte1p, blocks activation of Tem1p and consequently mitotic exit.

Cell Science at a Glance - inositides (p. 2207)

The attachment of lipid and phosphate groups to the inositol ring generates an astonishing array of molecules - confusing enough even before you try to get to grips with the nomenclature and the multitude of enzymes. In this issue’s Cell Science at a Glance, Stephen Shears and co-workers present a user-friendly summary of all the phosphoinositides, inositol phosphates and enzymatic reactions involved, which we hope will make this metabolic pathway a little less confusing.