Annual Review of Cell and Developmental Biology - Volume 14, 1998

The modification of proteins by chains of ubiquitin has long been known to mediate targeting of cytosolic and nuclear proteins for degradation by proteasomes. In this article, we discuss recent developments that reveal the involvement of ubiquitin in the degradation of proteins retained within the endoplasmic reticulum (ER) and in the internalization of plasma membrane proteins. Both luminal and transmembrane proteins retained in the ER are now known to be retrotranslocated into the cytosol in a process that involves ER chaperones and components of the protein import machinery. Once exposed to the cytosolic milieu, retro-translocated proteins are degraded by the proteasome, in most cases following polyubiquitination. There is growing evidence that both the ubiquitin-conjugating machinery and proteasomes may be associated with the cytosolic face of the ER membrane and that they could be functionally coupled to the process of retro-translocation. The ubiquitination of plasma membrane proteins, on the other hand, mediates internalization of the proteins, which in most cases is followed by lysosomal/vacuolar degradation. There is, however, a well-documented case of a plasma membrane protein (the c-Met receptor) for which ubiquitination results in proteasomal degradation. These recent findings imply that ubiquitin plays more diverse roles in the regulation of the fate of cellular proteins than originally anticipated.

Wnt genes encode a large family of secreted, cysteine-rich proteins that play key roles as intercellular signaling molecules in development. Genetic studies in Drosophila and Caenorhabditis elegans, ectopic gene expression in Xenopus, and gene knockouts in the mouse have demonstrated the involvement of Wnts in processes as diverse as segmentation, CNS patterning, and control of asymmetric cell divisions. The transduction of Wnt signals between cells proceeds in a complex series of events including post-translational modification and secretion of Wnts, binding to transmembrane receptors, activation of cytoplasmic effectors, and, finally, transcriptional regulation of target genes. Over the past two years our understanding of Wnt signaling has been substantially improved by the identification of Frizzled proteins as cell surface receptors for Wnts and by the finding that β-catenin, a component downstream of the receptor, can translocate to the nucleus and function as a transcriptional activator. Here we review recent data that have started to unravel the mechanisms of Wnt signaling.

The tight junction forms a regulated barrier in the paracellular pathway between epithelial and endothelial cells. This intercellular junction also demarcates the compositionally distinct apical and basolateral membranes. While the existence of a paracellular barrier in epithelia was hypothesized by physiologists over a century ago, the molecular characterization of the tight junction is a relatively new and rapidly expanding area of research. It is now recognized that the tight junction is comprised of at least nine peripheral and one integral membrane proteins. This complex includes members of a protein family related to tumor suppression and signal transduction, a rab protein, and a Ras target protein. The characteristics of, interactions between, and potential physiological roles of these proteins at the tight junction are discussed.

Recent studies showing that detergent-resistant membrane fragments can be isolated from cells suggest that biological membranes are not always in a liquid-crystalline phase. Instead, sphingolipid and cholesterol-rich membranes such as plasma membranes appear to exist, at least partially, in the liquid-ordered phase or a phase with similar properties. Sphingolipid and cholesterol-rich domains may exist as phase-separated “rafts” in the membrane. We discuss the relationship between detergent-resistant membranes, rafts, caveolae, and low-density plasma membrane fragments. We also discuss possible functions of lipid rafts in membranes. Signal transduction through the high-affinity receptor for IgE on basophils, and possibly through related receptors on other hematopoietic cells, appears to be enhanced by association with rafts. Raft association may also aid in signaling through proteins anchored by glycosylphosphatidylinositol, particularly in hematopoietic cells and neurons. Rafts may also function in sorting and trafficking through the secretory and endocytic pathways.

Many pathogens actively exploit the actin cytoskeleton during infection. This exploitation may take place during entry into mammalian cells after engagement of a receptor and/or as series of signaling events culminating in the engulfment of the microorganism. Although actin rearrangements are a common feature of most internalization events (e.g. entry of Listeria, Salmonella, Shigella, Yersinia, Neisseria, and Bartonella), bacterial and other cellular factors involved in entry are specific to each bacterium. Another step during which pathogens harness the actin cytoskeleton takes place in the cytosol, within which some bacteria (Listeria, Shigella, Rickettsia) or viruses (vaccinia virus) are able to move. Movement is coupled to a polarized actin polymerization process, with the formation of characteristic actin tails. Increasing attention has focused on this phenomenon due to its striking similarity to cellular events occurring at the leading edge of locomoting cells. Thus pathogens are convenient systems in which to study actin cytoskeleton rearrangements in response to stimuli at the plasma membrane or inside cells.

Metazoans contain multiple types of muscle cells that share several common properties, including contractility, excitability, and expression of overlapping sets of muscle structural genes that mediate these functions. Recent biochemical and genetic studies have demonstrated that members of the myocyte enhancer factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, serum response factor)-box transcription factors play multiple roles in muscle cells to control myogenesis and morphogenesis. Like other MADS-box proteins, MEF2 proteins act combinatorially through protein-protein interactions with other transcription factors to control specific sets of target genes. Genetic studies in Drosophila have also begun to reveal the upstream elements of myogenic regulatory hierarchies that control MEF2 expression during development of skeletal, cardiac, and visceral muscle lineages. Paradoxically, MEF2 factors also regulate cell proliferation by functioning as endpoints for a variety of growth factor-regulated intracellular signaling pathways that are antagonistic to muscle differentiation. We discuss the diverse functions of this family of transcription factors, the ways in which they are regulated, and their mechanisms of action.

Bioluminescence has evolved independently many times; thus the responsible genes are unrelated in bacteria, unicellular algae, coelenterates, beetles, fishes, and others. Chemically, all involve exergonic reactions of molecular oxygen with different substrates (luciferins) and enzymes (luciferases), resulting in photons of visible light (≈50 kcal). In addition to the structure of luciferan, several factors determine the color of the emissions, such as the amino acid sequence of the luciferase (as in beetles, for example) or the presence of accessory proteins, notably GFP, discovered in coelenterates and now used as a reporter of gene expression and a cellular marker. The mechanisms used to control the intensity and kinetics of luminescence, often emitted as flashes, also vary. Bioluminescence is credited with the discovery of how some bacteria, luminous or not, sense their density and regulate specific genes by chemical communication, as in the fascinating example of symbiosis between luminous bacteria and squid.

Signaling roles for phosphoinositides that involve their regulated hydrolysis to generate second messengers have been well characterized. Recent work has revealed additional signaling roles for phosphoinositides that do not involve their hydrolysis. PtdIns 3-P, PtdIns 3,4,5-P3, and PtdIns 4,5-P2 function as site-specific signals on membranes that recruit and/or activate proteins for the assembly of spatially localized functional complexes. A large number of phosphoinositide-binding proteins have been identified as the potential effectors for phosphoinositide signals. Common themes of localized signal generation and the spatially localized recruitment of effector proteins appear to underlie mechanisms employed in signal transduction, cytoskeletal, and membrane trafficking events.

Proteins that control mitochondrial dynamics in yeast are being identified at a rapid pace. These proteins include cytoskeletal elements that regulate organelle distribution and inheritance and several outer membrane proteins that are required to maintain the branched, mitochondrial reticulum. Interestingly, three of the high molecular weight GTPases encoded by the yeast genome are required for mitochondrial integrity and are potential regulators of mitochondrial branching, distribution, and membrane fusion. The recent finding that mtDNA mixing is restricted in the mitochondrial matrix has stimulated the hunt for the molecular machinery that anchors mitochondrial nucleoids in the organelle. Considering that many aspects of mitochondrial structure and behavior are strikingly similar in different cell types, the functional analyses of these yeast proteins should provide general insights into the mechanisms governing mitochondrial dynamics in all eukaryotes.

The actin cytoskeleton is a highly dynamic network composed of actin polymers and a large variety of associated proteins. The main functions of the actin cytoskeleton are to mediate cell motility and cell shape changes during the cell cycle and in response to extracellular stimuli, to organize the cytoplasm, and to generate mechanical forces within the cell. The reshaping and functions of the actin cytoskeleton are regulated by signaling pathways. Here we broadly review the actin cytoskeleton and the signaling pathways that regulate it. We place heavy emphasis on the yeast actin cytoskeleton.

The generation of isoforms via gene duplication and alternative splicing has been a valuable evolutionary tool for the creation of biological diversity. In addition to the formation of molecules with related but different functional characteristics, it is now apparent that isoforms can be segregated into different intracellular sites within the same cell. Sorting has been observed in a wide range of genes, including those encoding structural molecules, receptors, channels, enzymes, and signaling molecules. This results in the creation of intracellular compartments that (a) can be independently controlled and (b) have different functional properties. The sorting mechanisms are likely to operate at the level of both proteins and mRNAs. Isoform sorting may be an important consequence of the evolution of isoforms and is likely to have contributed to the diversity of functional properties within groups of isoforms.

A single plant produces several different types of leaves or leaf-like organs during its life span. This phenomenon, which is termed heteroblasty, is an invariant feature of shoot development but is also regulated by environmental factors that affect the physiology of the plant. Invariant patterns of heteroblastic development reflect global changes in the developmental status of the shoot, such as the progression from embryogenesis through juvenile and adult phases of vegetative development, culminating in the production of reproductive structures. Genes that regulate these phase-specific aspects of leaf identity have been identified by mutational analysis in both maize and Arabidopsis. These mutations have revealed that leaf production is regulated independently of leaf identity, implying that the identity of a leaf at a particular position on the shoot may depend on when the leaf was initiated in relation to a temporal program of shoot development.

Regulation of translation initiation is a central control point in animal cells. We review our current understanding of the mechanisms of regulation, drawing particularly on examples in which the biological consequences of the regulation are clear. Specific mRNAs can be controlled via sequences in their 5′ and 3′ untranslated regions (UTRs) and by alterations in the translation machinery. The 5′UTR sequence can determine which initiation pathway is used to bring the ribosome to the initiation codon, how efficiently initiation occurs, and which initiation site is selected. 5′UTR-mediated control can also be accomplished via sequence-specific mRNA-binding proteins. Sequences in the 3′ untranslated region and the poly(A) tail can have dramatic effects on initiation frequency, with particularly profound effects in oogenesis and early development. The mechanism by which 3′UTRs and poly(A) regulate initiation may involve contacts between proteins bound to these regions and the basal translation apparatus. mRNA localization signals in the 3′UTR can also dramatically influence translational activation and repression. Modulations of the initiation machinery, including phosphorylation of initiation factors and their regulated association with other proteins, can regulate both specific mRNAs and overall translation rates and thereby affect cell growth and phenotype.

Cells respond to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing transcription of genes encoding ER resident proteins. The information is transmitted from the ER lumen to the nucleus by an intracellular signaling pathway called the unfolded protein response (UPR). Recent work has shown that this signaling pathway utilizes several novel mechanisms, including translational attenuation and a regulated mRNA splicing step. In this review we aim to integrate these recent advances with current knowledge about maintenance of ER composition and abundance.

Actin filaments (thin filaments) are polymerized to strikingly uniform lengths in striated muscle sarcomeres. Yet, actin monomers can exchange dynamically into thin filaments in vivo, indicating that actin monomer association and dissociation at filament ends must be highly regulated to maintain the uniformity of filament lengths. We propose several hypothetical mechanisms that could generate uniform actin filament length distributions and discuss their application to the determination of thin filament length in vivo. At the Z line, titin may determine the minimum extent and tropomyosin the maximum extent of thin filament overlap by regulating α-actinin binding to actin, while a unique Z filament may bind to capZ and regulate barbed end capping. For the free portion of the thin filament, we evaluate possibilities that thin filament components (e.g. nebulin or the tropomyosin/troponin polymer) determine thin filament lengths by binding directly to tropomodulin and regulating pointed end capping, or alternatively, that myosin thick filaments, together with titin, determine filament length by indirectly regulating tropomodulin's capping activity.