By regulating axonal pathfinding, dendritic growth and arborization, and specification of neurotransmitter subtype, calcium-mediated signaling produces a diverse array of neuronal subtypes.
The development of the nervous system involves the generation of a stunningly diverse array of neuronal subtypes that enable complex information processing and behavioral outputs. Deciphering how the nervous system acquires and interprets information and orchestrates behaviors will be greatly enhanced by the identification of distinct neuronal circuits and by an understanding of how these circuits are formed, changed, and/or maintained over time. Addressing these challenging questions depends in part on the ability to accurately identify and characterize the unique neuronal subtypes that comprise individual circuits. Distinguishing characteristics of neuronal subgroups include but are not limited to neurotransmitter phenotype, dendritic morphology, the identity of synaptic partners, and the expression of constellations of subgroup-specific proteins, including ion channel subtypes.
Thrombospondins are glycoproteins that associate with the extracellular matrix and have roles in cell signaling and tissue remodeling.
Thrombospondins are evolutionarily conserved, calcium-binding glycoproteins that undergo transient or longer-term interactions with other extracellular matrix components. They share properties with other matrix molecules, cytokines, adaptor proteins, and chaperones, modulate the organization of collagen fibrils, and bind and localize an array of growth factors or proteases. At cell surfaces, interactions with an array of receptors activate cell-dependent signaling and phenotypic outcomes. Through these dynamic, pleiotropic, and context-dependent pathways, mammalian thrombospondins contribute to wound healing and angiogenesis, vessel wall biology, connective tissue organization, and synaptogenesis. We overview the domain organization and structure of thrombospondins, key features of their evolution, and their cell biology. We discuss their roles in vivo, associations with human disease, and ongoing translational applications. In many respects, we are only beginning to appreciate the important roles of these proteins in physiology and pathology.
Cargo transport and membrane fusion within the Golgi is mediated by four SNARE complexes. SM (Sec1p/Munc18) proteins and tethering factors (e.g., golgins) control the spatiotemporal activity and assembly of these SNARE complexes.
Antero- and retrograde cargo transport through the Golgi requires a series of membrane fusion events. Fusion occurs at the cis- and trans-side and along the rims of the Golgi stack. Four functional SNARE complexes have been identified mediating lipid bilayer merger in the Golgi. Their function is tightly controlled by a series of reactions involving vesicle tethering and SM proteins. This network of protein interactions spatially and temporally determines the specificity of transport vesicle targeting and fusion within the Golgi.
The kinase MEK1 stimulates disassembly of the Golgi at mitosis to facilitate its partitioning into daughter cells. SNARE proteins then promote fusion of the resulting vesicles to regenerate a functional Golgi apparatus at the end of mitosis.
The Golgi is an essential membrane-bound organelle in the secretary pathway of eukaryotic cells. In mammalian cells, the Golgi stacks are integrated into a continuous perinuclear ribbon, which poses a challenge for the daughter cells to inherit this membrane organelle during cell division. To facilitate proper partitioning, the mammalian Golgi ribbon is disassembled into vesicles in early mitosis. Following segregation into the daughter cells, a functional Golgi is reformed. Here we summarize our current understanding of the molecular mechanisms that control the mitotic Golgi disassembly and postmitotic reassembly cycle in mammalian cells.
In female germ cells, the inactive X chromosome is reactivated before meiosis and thereafter remains active. In contrast, the X chromosome in males is inactivated during meiosis, and silencing is largely maintained during spermiogenesis.
The sex chromosomes play a highly specialized role in germ cell development in mammals, being enriched in genes expressed in the testis and ovary. Sex chromosome abnormalities (e.g., Klinefelter [XXY] and Turner [XO] syndrome) constitute the largest class of chromosome abnormalities and the commonest genetic cause of infertility in humans. Understanding how sex-gene expression is regulated is therefore critical to our understanding of human reproduction. Here, we describe how the expression of sex-linked genes varies during germ cell development; in females, the inactive X chromosome is reactivated before meiosis, whereas in males the X and Y chromosomes are inactivated at this stage. We discuss the epigenetics of sex chromosome inactivation and how this process has influenced the gene content of the mammalian X and Y chromosomes. We also present working models for how perturbations in sex chromosome inactivation or reactivation result in subfertility in the major classes of sex chromosome abnormalities.
Oocyte meiotic arrest, maturation, and egg activation are regulated by both conserved and unique molecular pathways in mammals, Caenorhabditis elegans, and Drosophila. There are striking parallels in mouse and C. elegans for the role of somatic cells in oocyte maturation. In Drosophila and C. elegans specific kinases act uniquely to control the oocyte to zygote transition.
Production of functional eggs requires meiosis to be coordinated with developmental signals. Oocytes arrest in prophase I to permit oocyte differentiation, and in most animals, a second meiotic arrest links completion of meiosis to fertilization. Comparison of oocyte maturation and egg activation between mammals, Caenorhabditis elegans, and Drosophila reveal conserved signaling pathways and regulatory mechanisms as well as unique adaptations for reproductive strategies. Recent studies in mammals and C. elegans show the role of signaling between surrounding somatic cells and the oocyte in maintaining the prophase I arrest and controlling maturation. Proteins that regulate levels of active Cdk1/cyclin B during prophase I arrest have been identified in Drosophila. Protein kinases play crucial roles in the transition from meiosis in the oocyte to mitotic embryonic divisions in C. elegans and Drosophila. Here we will contrast the regulation of key meiotic events in oocytes.
Hundreds of different lipid species are present in eukaryotic cell membranes. Some of them aggregate with specific membrane proteins to form specialized domains that concentrate and control cellular trafficking and signaling events.
Cell membranes are composed of a lipid bilayer, containing proteins that span the bilayer and/or interact with the lipids on either side of the two leaflets. Although recent advances in lipid analytics show that membranes in eukaryotic cells contain hundreds of different lipid species, the function of this lipid diversity remains enigmatic. The basic structure of cell membranes is the lipid bilayer, composed of two apposing leaflets, forming a two-dimensional liquid with fascinating properties designed to perform the functions cells require. To coordinate these functions, the bilayer has evolved the propensity to segregate its constituents laterally. This capability is based on dynamic liquid–liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. This principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to focus and regulate membrane bioactivity. Here we will review the emerging principles of membrane architecture with special emphasis on lipid organization and domain formation.
For both naked and enveloped viruses, viral entry, genome replication, and egress involve specific interactions with the membranes of a susceptible host cell. Their characterization has led to the discovery of lipid-active compounds as potential antiviral drugs.
Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.
PD is caused by proteostasis dysfunction, specifically the misfolding and self-association of aberrant α-synuclein. ALS may have several causes, including protein mislocalization and faulty RNA quality control.
Parkinson’s disease (PD) is a movement disorder that afflicts over one million in the U.S.; amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) is less prevalent but also has a high incidence. The two disorders sometimes present together, making a comparative study of interest. Both ALS and PD are neurodegenerative diseases, and are characterized by the presence of intraneuronal inclusions; however, different classes of neurons are affected and the primary protein in the inclusions differs between the diseases, and in some cases is different in distinct forms of the same disease. These observations might suggest that the more general approach of proteostasis pathway alteration would be a powerful one in treating these disorders. Examining results from human genetics and studies in model organisms, as well as from biochemical and biophysical characterization of the proteins involved in both diseases, we find that most instances of PD can be considered as arising from the misfolding, and self-association to a toxic species, of the small neuronal protein α-synuclein, and that proteostasis strategies are likely to be of value for this disorder. For ALS, the situation is much more complex and less clear-cut; the available data are most consistent with a view that ALS may actually be a family of disorders, presenting similarly but arising from distinct and nonoverlapping causes, including mislocalization of some properly folded proteins and derangement of RNA quality control pathways. Applying proteostasis approaches to this disease may require rethinking or broadening the concept of what proteostasis means.
Argonaute proteins lie at the heart of all RNAi-related effector complexes. Their PAZ domain recognizes small RNA targets; the PIWI domain is related to RNase H and responsible for target cleavage.
RNAi has existed at least since the divergence of prokaryotes and eukaryotes. This collection of pathways responds to a diversity of “abberant” RNAs and generally silences or eliminates genes sharing sequence content with the silencing trigger. In the canonical pathway, double-stranded RNAs are processed into small RNAs, which guide effector complexes to their targets by complementary base pairing. Many alternative routes from silencing trigger to small RNA are continuously being uncovered. Though the triggers of the pathway and the mechanisms of small RNA production are many, all RNAi-related mechanisms share Argonaute proteins as the heart of their effector complexes. These can act as self-contained silencing machines, binding directly to small RNAs, carrying out homology-based target recognition, and in some cases cleaving targets using an endogenous nuclease domain. Here, we discuss the diversity of Argonaute proteins from a structural and functional perspective.
Decades of extensive biochemical and biophysical research have outlined the mechanism of translation. Rich structural studies have provided detailed snapshots of the translational machinery at all stages of the translation cycle. However the relationship between structural dynamics, composition, and function remains unknown. The heterogeneous and asynchronous nature of translation hinders elucidation of its underlying molecular mechanisms by conventional methods. Single-molecule approaches unsusceptible to these complications have led to the first glances at both compositional and conformational dynamics on the ribosome. These experiments provide the necessary link between static structure and mechanism, often providing new perspectives. Here we review recent advances in the field and their relationship to structural and biochemical data.
The endoplasmic reticulum (ER) is the site of synthesis for nearly one-third of the eukaryotic proteome and is accordingly endowed with specialized machinery to ensure that proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by ER-associated degradation (ERAD), a complex process through which folding-defective proteins are selected and ultimately degraded by the ubiquitin-proteasome system. ERAD proceeds through four tightly coupled steps involving substrate selection, dislocation across the ER membrane, covalent conjugation with polyubiquitin, and proteasomal degradation. The ERAD machinery shows a modular organization with central ER membrane-embedded ubiquitin ligases linking components responsible for recognition in the ER lumen to the ubiquitin-proteasome system in the cytoplasm. The core ERAD machinery is highly conserved among eukaryotes and much of our basic understanding of ERAD organization has been derived from genetic and biochemical studies of yeast. In this article we discuss how the core ERAD machinery is organized in mammalian cells.
Changes in intracellular Ca2+ are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca2+ signal is a reflection of the source of Ca2+ (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca2+ entry may be detected optically as graded elevations in intracellular Ca2+, junctional Ca2+ transients, Ca2+ flashes, or Ca2+ sparklets, whereas release of Ca2+ from intracellular stores may manifest as Ca2+ sparks, Ca2+ puffs, or Ca2+ waves. These diverse Ca2+ signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).
Ca2+ signals in smooth muscle include Ca2+ sparks (mediated by RyRs), Ca2+ puffs (IP3Rs), Ca2+ waves (IP3Rs/RyRs), junctional Ca2+ transients (P2X1Rs), Ca2+ flashes (VDCCs), and Ca2+ sparklets (VDCCs).
Integrin-based adhesion has served as a model for studying the central role of adhesion in migration. In this article, we outline modes of migration, both integrin-dependent and -independent in vitro and in vivo. We next discuss the roles of adhesion contacts as signaling centers and linkages between the ECM and actin that allows adhesions to serve as traction sites. This includes signaling complexes that regulate migration and the interplay among adhesion, signaling, and pliability of the substratum. Finally, we address mechanisms of adhesion assembly and disassembly and the role of adhesion in cellular polarity.
During cell migration, integrin-mediated adhesions dynamically form and turn over. The polarized assembly and disassembly of adhesions are essential for optimum cell speed and directional persistence.
The Golgi is essential for processing proteins and sorting them, as well as plasma membrane components, to their final destinations. Not surprisingly, this organelle, a major compartment of the secretory pathway, is an important venue for regulating many aspects of development in both invertebrates and vertebrates. Through its role as a site for protein cleavage and glycosylation as well as through changes in its spatial organization and secretory trafficking, the Golgi exerts highly specific effects on cellular differentiation and morphogenesis by spatially and temporally constraining developmental pathways.
Developmental pathways in invertebrates and vertebrates are controlled in part by specific protein modifications and trafficking events at the Golgi. However, different species and cell types use Golgi in different ways to influence development.
The Golgi factory receives custom glycosylates and dispatches its cargo to the correct cellular locations. The process requires importing donor substrates, moving the cargo, and recycling machinery. Correctly glycosylated cargo reflects the Golgi's quality and efficiency. Genetic disorders in the specific equipment (enzymes), donors (nucleotide sugar transporters), or equipment recycling/reorganization components (COG, SEC, golgins) can all affect glycosylation. Dozens of human glycosylation disorders fit these categories. Many other genes, with or without familiar names, well-annotated pedigrees, or likely homologies will join the ranks of glycosylation disorders. Their broad and unpredictable case-by-case phenotypes cross the traditional medical specialty boundaries. The gene functions in patients may be elusive, but their common feature may include altered glycosylation that provide clues to Golgi function. This article focuses on a group of human disorders that affect protein or lipid glycosylation. Readers may find it useful to generalize some of these patient-based, translational observations to their own research.
The Golgi glycosylates and sorts intracellular protein and lipid cargos. Impaired performance by mutated Golgi resident proteins creates severe and highly variable pathologies.
Small noncoding RNAs have emerged as potent regulators of gene expression, especially in the germline. We review the biogenesis and regulatory function of three major small noncoding RNA pathways in the germline: The small interfering RNA (siRNA) pathway that leads to the degradation of target mRNAs, the microRNA (miRNA) pathway that mostly represses the translation of target mRNAs, and the newly discovered Piwi-interacting RNA (piRNA) pathway that appears to have diverse functions in epigenetic programming, transposon silencing, and the regulation of mRNA translation and stability. The siRNA and miRNA pathways are present in the germline as well as many somatic tissues, whereas the piRNA pathway is predominantly confined to the germline. Investigation of the three small RNA pathways has started to reveal a new dimension of gene regulation with defining roles in germline specification and development.
The small interfering RNA (siRNA) pathway leads to the degradation of target mRNAs; the microRNA (miRNA) pathway mainly represses the translation of target mRNAs; and the Piwi-interacting RNA (piRNA) pathway may function in epigenetic programming, transposon silencing, and mRNA regulation.
Translational control of specific mRNAs is a widespread mechanism of gene regulation, and it is especially important in pattern formation in the oocytes of organisms in which the embryonic axes are established maternally. Drosophila and Xenopus have been especially valuable in elucidating the relevant molecular mechanisms. Here, we comprehensively review what is known about translational control in these two systems, focusing on examples that illustrate key concepts that have emerged. We focus on protein-mediated translational control, rather than regulation mediated by small RNAs, as the former appears to be predominant in controlling these developmental events. Mechanisms that modulate the ability of the specific mRNAs to be recruited to the ribosome, that regulate polyadenylation of specific mRNAs, or that control the association of particular mRNAs into translationally inert ribonucleoprotein complexes will all be discussed.
Protein-mediated translational regulation has been intensively studied in Drosophila and Xenopus oocytes. Some mechanisms (e.g., cytoplasmic polyadenylation-induced translation) also occur in somatic cells and may be common to all metazoans.
Despite their compositional complexity, lipidomes comprise a large number of isobaric species that cannot be distinguished by conventional low resolution mass spectrometry and therefore in-depth MS/MS analysis was required for their accurate quantification. Here we argue that the progress in high resolution mass spectrometry is changing the concept of lipidome characterization. Because exact masses of isobaric species belonging to different lipid classes are not necessarily identical, they can now be distinguished and directly quantified in total lipid extracts. By streamlining and simplifying the molecular characterization of lipidomes, high resolution mass spectrometry has developed into a generic tool for cell biology and molecular medicine.
High-resolution mass spectrometry streamlines the shotgun analysis of total lipid extracts. It can distinguish and quantify isobaric lipid species and has potential to become the “gold standard” lipidomics methodology.
Ever since it was discovered that biological membranes have a core of a bimolecular sheet of lipid molecules, lipid bilayers have been a model laboratory for investigating physicochemical and functional properties of biological membranes. Experimental and theoretical models help the experimental scientist to plan experiments and interpret data. Theoretical models are the theoretical scientist’s preferred toys to make contact between membrane theory and experiments. Most importantly, models serve to shape our intuition about which membrane questions are the more fundamental and relevant ones to pursue. Here we review some membrane models for lipid self-assembly, monolayers, bilayers, liposomes, and lipid–protein interactions and illustrate how such models can help answering questions in modern lipid cell biology.
Questions in modern lipid cell biology have been answered by membrane models such as SUVs, LUVs, and GUVs—closed lipid bilayers that are used to study effects of membrane permeability barriers and curvature. However, new membrane models are needed for answering pressing questions regarding membrane structure and function in nonequilibrium states.
Genetically engineered mice are critical experimental models for the study of breast cancer biology. Transgenic mice, employing strong mammary epithelial promoters to drive oncogenes, develop carcinomas with phenotypes corresponding to the molecular pathway activated. Gene-targeted (knockout) mice, in which tumor suppressors are deleted, develop mammary neoplasms with phenotypes primarily including patterns seen in spontaneous mouse mammary tumors, albeit at higher rates. Improved genetic engineering, using inducible gene expression, somatic gene transduction, conditional alleles, and crossbreeding for combined/compound genetic engineering yields precise molecular models with exquisite experimental control and phenotypes with comparative pathologic validity. Mammary gland transplantation technology adds a practical and validated method for assessing biologic behavior of selected mammary tissues. Overall, the many mouse models available are a rich resource for experimental biology with phenocopies of breast cancer subtypes, and a variety of practical advantages. The challenge is matching the model to the experimental question.
Human cancers are genotypically, phenotypically, and behaviorally heterogeneous. However, specific molecular alterations (e.g., Erbb2 amplification, found in 25% of human breast cancers) have been successfully modeled in mice.
The endoplasmic reticulum (ER) is the primary site for synthesis and folding of secreted and membrane-bound proteins. Proteins are translocated into ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to assist in proper folding. Properly folded proteins traffic from the ER to the Golgi apparatus; misfolded proteins are targeted to degradation. Unfolded protein response (UPR) is a highly regulated intracellular signaling pathway that prevents accumulation of misfolded proteins in the ER lumen. UPR provides an adaptive mechanism by which cells can augment protein folding and processing capacities of the ER. If protein misfolding is not resolved, the UPR triggers apoptotic cascades. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintain cellular homeostasis and determine cell fate under various pathophysiological conditions. Regulated Ca2+ transfer from the ER to the mitochondria is important in maintaining control of prosurvival/prodeath pathways. We discuss the signaling/communication between the ER and mitochondria and focus on the role of the mitochondrial permeability transition pore in these complex processes.
If protein misfolding in the ER is not resolved by the unfolded protein response (UPR), apoptosis is triggered. The is regulated by Ca2+ transfer from the ER to the mitochondria.