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Organizational Cell Biology: An Overview
Organizational Cell Biology: An Overview EL Elson, Washington University School of Medicine, St. Louis, MO, USA G Raposo, Institut Curie, Paris, France PA Gleeson, The University of Melbourne, Melbourne, VIC, Australia A Akhmanova, Utrecht University, Kruytgebouw, Utrecht, The Netherlands T Yoshimori, Osaka University, Suita, Osaka, Japan PD Stahl, Washington University School of Medicine, St. Louis, MO, USA B Goud, Institut Curie, Paris, France r 2016 Elsevier Inc. All rights reserved.
The cell is the building block of life and, as written by Lewis Wolpert (1999), can be considered as the evolution’s most magnificent achievement. Cells, in particular animal cells (plants cells being surrounded by a wall that imposes a constraint), display all kinds of shapes and sizes, and many different types are present in the same living organism (for instance, more than 350 types of cells have been described in humans). Besides this huge diversity, all cells share the same fundamental feature, i.e., the capacity to behave as stable functional units. Such remarkable functional stability allows them to resist change over time and to respond to threats. The cell functional stability relies on its organization in compartments or organelles, which are themselves organized in cellular space by the cell cytoskeleton. Still this organization is not static and the development of live cell imaging approaches in the last 20 years has illustrated the extraordinary dynamics of cell compartments and the multiple connections that exist among them. On the other hand, the loss of stability at the cell level leads to a pathological state, a remarkable example being cancer progression. A loss of stability can also be provoked by pathogens and toxins, which in many cases, can subvert cellular functions to their own benefit. Part II of the Encyclopedia is divided into five sections that describe the range of imaging approaches developed to date to understand the cell organization (see Section Imaging the Cell), the principle organelles (see Section Organelles), the communications between organelles (see Section Interorganellar Communication), how cell architecture is maintained (see Section Cytoskeleton and Motors), and how cell organization can be altered by pathogens (see Section Intracellular Infectiology).
Imaging the Cell The potential of optical methods to acquire a dynamic view of molecules, cells, tissues, and organisms is extraordinary indeed, microscopy is the Dean of Cell Biology. Both light and electron microscopy have moved cell biology into a new era. Yet subcellular structures and organelles, their specific morphological and compositional characteristics, their intimate details and possible connections can be seen only by high-resolution transmission and scanning electron microscopy (TEM and SEM, respectively). TEM has been used for more than 70 years to investigate the ultrastructure of cells and tissues in normal and pathological situations. In more recent times methods have been developed that allow for the analysis of molecular complexes, fine details and 3D analysis of cells,
Encyclopedia of Cell Biology, Volume 2
tissues, small organisms, and subcellular structures including the localization of cellular components with an intraorganellar context. Single-particle cryoEM allows for a near-atomic (B3– 4 Å) resolution of structures or macromolecular complexes such as virus particles, molecular machineries, and membrane proteins. Importantly, these can be viewed in close to native state, without contrasting agents and after vitrification by Cryo-EM (Single-Particle CryoEM of Macromolecular Complexes). Scanning EM has a lower resolution than TEM but it is the only method that permits one to delineate the surface of cells and small organisms and to appreciate structural details thanks to its depth of field and three-dimensional (3D) surface imaging characteristics (Scanning Electron Microscopy in Cell Biology). These classical SEM methods are powerful and have been improved lately by developments in electron microscope technology permitting visualization of isolated organelles and large complexes such as nuclear pores (Scanning Electron Microscopy in Cell Biology). Unfortunately, they do not provide 3D information on the internal characteristics of cells, tissues, and organisms. Over the past few years this limitation has been surpassed by combining SEM imaging with the use of a microtome or a focused ion beam (FIB) that progressively removes sections from the top of the object under study thus uncovering fine details of the interior structures. The acquired images can be stacked and a 3D model can be generated to appreciate the internal organization of a specimen that may be composed of several cells (Imaging Cellular Architecture with 3D SEM). In a very complementary manner and with a higher resolution, Electron Tomography allows one to resolve the 3D organization of molecular and subcellular structures by TEM of plastic embedded sections of cells and tissues and CryoTEM of unstained, vitrified specimens (Electron Tomography). Visualization of molecules within their intracellular and intraorganellar contexts can be achieved using electron dense probes that can be viewed by TEM. Localization methods are most generally based on the recognition of molecules by specific antibodies (Immuno-EM). Such methods combine localization and preservation of ultrastructure and permit discrimination of seemingly identical compartments by their molecular composition in a quantitative manner (Immunoelectron Microscopy: High-Resolution Immunocytochemistry). For many years fluorescence has been the dominant optical property used to study structural and functional properties of cells. Its advantageous qualities include chemical selectivity, i.e., the ability to measure specifically labeled molecular species at very low concentration in the presence of high concentrations of unlabeled molecules, relative ease and speed of measurement, high spatial resolution when used with a
doi:10.1016/B978-0-12-394447-4.20103-X
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Organizational Cell Biology: An Overview
confocal or 2-photon fluorescence microscope and, when used judiciously, lack of interference with normal cell function. The articles in this section of the Encyclopedia provide an introduction to methods that exploit recent advances in microscopy and labeling technology that have opened a new golden age of applications of fluorescence to cell biology. Several of the articles describe methods that enable imaging resolution beyond the Abbe/Rayleigh limit in the range of 200–300 nm for visible light. The oldest of these is total internal reflection microscopy (TIRFM) that provides sub-diffraction optical resolution along the optical axis and so has been a mainstay of studies of dynamic processes of single molecules bound to glass substrates. TIRFM does not, however, improve lateral resolution (Total Internal Reflection Fluorescence Microscopy). Localization methods such as PALM, STORM, and FPALM assemble high-resolution images of macromolecular structures by summing the contributions of component fluorophores whose individual positions are determined with accuracies near 10 nm (Super Resolution Fluorescence Localization Microscopy). As described in article Super-Resolution Light Microscopy: Stimulated Emission Depletion and Ground-State Depletion, STED microscopy is scanning superresolution approach based on restricting the radius of the excitation light to sub-diffraction dimensions. STED has the advantage of acquiring fluorescence signals rapidly enough to be useful in studies of dynamic molecular behavior, for example, via fluorescence correlation spectroscopy (FCS). Structured illumination microscopy (SIM) breaks the diffraction limit by imposing a periodic spatial pattern on the excitation light. Although SIM does not match the resolution of STED and localization methods, it has other advantages of flexibility, relative ease of use and high throughput (Structured Illumination Microscopy). At a higher structural level intravital microscopy (Intravital Microscopy in Mammalian Organisms: From Tissue Physiology to Cell Biology) is used to investigate tissue and cell architecture in living animals and cells. This method has recently benefitted from applications of molecular genetics and improvements in microscopy and fluorescent probes. Fluorescence lifetimes are sensitive to the environment of the emitting fluorophore. Therefore, fluorescence lifetime imaging microscopy (FLIM) provides a unique and popular method for enhancing the contrast of fluorescence images to reveal regional differences in a variety of cellular properties and functions, for example, metabolism, ionic concentrations, and molecular composition (Fluorescence Lifetime Imaging – Applications and Instrumental Principles). Optogenetics (Optogenetics) uses light to trigger molecular functions, and thereby to control activities in living cells. This powerful new approach has mainly been used to control cellular excitability through regulating ion channel function. Although FCS was invented in the 1970s, many variations and extensions have been developed, since then continue to be valuable tools to investigate dynamic molecular processes in cells. The family of FCS and closely related photobleaching (FRAP (fluorescence recovery after photobleaching)) methods yield important information about molecular transport as well as rates and extents of molecular interactions (Fluorescence Correlation Spectroscopy: A Tool for Measuring Dynamic and Equilibrium Properties of Molecules in Cells). While FCS and FRAP rely on dynamic changes in the numbers of fluorescent molecules
detected within a small measurement area that can be no smaller than the optical resolution limit to measure transport rates, single-particle tracking extended by current localization microscopy methods allows measurement of the trajectories of individual molecules with precision in the range of 10 s of nm (High-Speed Localization Microscopy and Single-Particle Tracking). This approach allows a unique analysis of the mechanisms of motion of the tracked molecules. Finally, the use of genetically encoded fluorescent probes coupled with live cell imaging opens a window of opportunity to view cellular events in spectacular detail (Genetically Encoded Fluorescent Probes and Live Cell Imaging).
Organelles Acquisition of diverse intracellular compartments was truly revolutionary epoch in the long history of cellular evolution. Each compartment, known as an organelle, is enclosed by membrane, meaning that cells have a vast area of membrane surface at their disposal, in addition to the plasma membrane surrounding cells, which can act as a platform for biochemical reactions that sustain cellular activities. In addition, the interior space of organelles (the luminal space) sequestered by their limiting membranes also enables cells to perform distinct reactions efficiently. Indeed, organelles contain their own characteristic set of proteins and other molecules to achieve functions unique to each. Study of organelles is undoubtedly one of major streams in cell biology, which started when Robert Brown observed a small dark structure inside plant cells and named it ‘nucleus’ in 1831. This seminal discovery was followed by identification (or naming) of mitochondria by C. Benda and the Golgi apparatus by C. Golgi in the same year 1898, and lysosomes by C. de Duve in 1955. An enormous amount of research from these times has revealed the amazing ‘true colors’ of organelles, but we have not yet completely understood their function and structure; study is still ongoing. This section of the encyclopedia comprehensively covers major organelles including recently discovered and cell-type-specific organelles. Organization of the nucleus, a center for genome storage and for controlling its expression, is introduced in the article Nuclear Organization (Nuclear Structure and Dynamics). There is macromolecular traffic between nucleus and the cytoplasm via the nuclear pore, which is large protein complex inserted into the nuclear double membrane, as discussed in the article Nuclear Pores. Van Anken and Sitia (The Endoplasmic Reticulum) describe the multifunctions of the endoplasmic reticulum; synthesis of lipids and proteins, Caþþ ion storage, signal integration, and interaction with other organelles. Otera and Mihara (Mechanisms and Functions of Mitochondrial Dynamics) focus on mitochondria dynamics; these cellular power plants are highly dynamic and fusion/fission of their membranes play a pivotal physiological role. Cargo internalized into cells from outside is transferred sequentially along the endocytic pathway consisting of early endosomes (Early Endosomal Compartments; Endocytosis of Cargo Proteins: LDL), late endosomes (The Late Endosome), and lysosomes (Conventional and Secretory Lysosomes). Lysosomes are final destination in the pathway, but can fuse with plasma membrane under certain
Organizational Cell Biology: An Overview
conditions (secretory lysosomes), as discussed in the article Conventional and Secretory Lysosomes. In addition, endosomes also function as a platform of signaling, as described in the article Signaling from Endosomes. Membrane traffic from the cytoplasm to lysosomes also exists (the autophagic pathway), which is mediated by autophagosomes (At the Center of Autophagy: Autophagosomes). On the other hand, lipids and proteins synthesized in the endoplasmic reticulum are transported to the Golgi (Golgi and TGN) via intermediate compartments (Intermediate Compartment: A Sorting Station between the Endoplasmic Reticulum and the Golgi Apparatus) to be sorted into each destination. Peroxisomes functioning in oxygen metabolism and fatty acid degradation (Peroxisomes) and lipid droplets specialized for lipid storage and metabolism (Lipid Droplets) are also discussed. Wauben (Extracellular Vesicles) discusses the most-recently discovered organelles, extracellular vesicles, which are released from cells and have diverse important physiological functions. While the abovementioned organelles are ubiquitous in most cell types, there are some cell-type-specific organelles. Only plants and algae have chloroplasts for photosynthesis (Plastids: The Anabolic Factories of Plant Cells). Nerve cells in animals possess synaptic vesicles for neurotransmission, as described in the article Synaptosomes and Synaptic Vesicles. Lastly, Fukuda (LysosomeRelated Organelles) discuss a group of organelles having very specialized functions, for example, pigmentation derived from lysosomes. These lysosome-related organelles exist in various highly differentiated cells.
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multiple pathways from the trans-Golgi network to the cell surface and constitutive and regulated post-Golgi transport are discussed in the articles Post-Golgi Transport – Cargo, Carriers, and Pathways and Regulated versus Constitutive Secretion – A Major Form of Intercellular Communication. Endocytosis is also an important element of interorganellar transport, and this section discusses both the clathrin-mediated and non-clathrinmediated pathways in the articles Clathrin and ClathrinDependent Endocytosis and Clathrin Independent Endocytosis. Transport along the endosomal–lysosomal pathway is covered in the article Endosome to Lysosome Transport, and pathways that carry cargo along the retrograde transport route from the endosomes to the Golgi are discussed in the article Retrograde Transport. Machinery components of vesicle formation and vesicle fusion are essential for the regulation of these transport pathways, and in particular include small G proteins (Rabs of the Endosomal Recycling Pathway; Rabs and Other G Proteins), phosphoinositides (Interorganeller Communication: Role of Phosphoinositides in Membrane Traffic), Bar domain proteins (BAR Domains and BAR Domain Superfamily Proteins), SNAREs (SNAREs: Membrane Fusion and Beyond), ESCRTs (ESCRTing around the Cell), exocyst and tethering factors (Vesicle Tethers) and components of the vesicle coats that interact with cargo, such as adaptor complexes (Adaptor Proteins: Inter-Organelle Traffic Controllers) and retromer (The Retromer Complex). And finally also included are pathways of cell secretion that are independent of the pathway involving the ER and the Golgi, referred to as unconventional protein secretion (Unconventional Protein Secretion: Fibroblast Growth Factor 2 and Interleukin-1β as Examples).
Interorganellar Communication The compartmental organization of the eukaryotic cell is essential for the development of multicellular organisms, for the organization of tissues and organs, and the coordination of physiological functions. The molecular processes which regulate the biogenesis of membrane compartments and the mechanisms by which information is shuttled back and forth between the different intracellular compartments represents fundamental processes in all eukaryotic cells. The pioneering work by Palade and colleagues, which integrated EM technologies with biochemical approaches, laid the foundation for understanding organelle dynamics and interorganellar communication. Subsequent work over the past 3–4 decades has revealed many of the molecular mechanisms which regulate these processes. This section focuses on the molecular mechanisms underlying membrane transport pathways. ER export and ER stress responses are now well-understood and discussed in the articles ER–Golgi Transport and Endoplasmic Reticulum Stress in Disease. Golgi organization and transport is a complex field that has invoked a variety of ingenious approaches in an attempt to define the underlying mechanisms of intracellular transport, as discussed in the article Intra-Golgi Transport. The Golgi is also the site for the glycosylation, and the biosynthesis and role of N-linked glycans and O-glycans in cell biology and development that are discussed in the articles N-Linked Glycans (N-Glycans) and Extracellular O-Glycans, respectively. An emerging field arises from the appreciation that signaling controls transport along the constitutive secretion pathway (Regulation of the Secretory Pathway). Post-Golgi transport involves
Cytoskeleton and Motors The ability of cells to regulate their shape, organize their components, move, and divide depends on the networks of filaments collectively called the cytoskeleton. In contrast to the human skeleton, which remodels slowly, most cytoskeletal filaments are dynamic, because they are composed of polymers of small building blocks that can rapidly assemble and disassemble, depending on the cellular needs. Growth and depolymerization of cytoskeletal filaments can generate forces that can drive cell protrusion and promote chromosome separation during cell division. Moreover, some cytoskeletal filaments can serve as rails for the movement of motor proteins, which use the energy released by ATP hydrolysis to perform mechanical work. The activity of such motors is responsible for the locomotion of single cells, cell contraction and intracellular transport of membrane organelles, RNA, and protein complexes. This section describes the composition, structure, and dynamics of the major eukaryotic cytoskeletal filaments: actin (Actin Assembly Dynamics and Its Regulation in Motile and Morphogenetic Processes), microtubules (Microtubules and Microtubule-Associated Proteins (MAPs)), intermediate filaments (Intermediate Filaments), and septins (Septins: Cytoskeletal Filaments with Structural and Regulatory Functions). A separate article is dedicated to the structurally and functionally diverse cytoskeletal networks present in bacteria and archaea (Bacterial and Archaeal Cytoskeletons). Subsequent articles discuss the molecular mechanisms, motile properties,
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Organizational Cell Biology: An Overview
and functions of the major cytoskeletal motor families. These include the actin-based motors, myosins (Myosins), and the microtubule-based motors, kinesins (Kinesin Superfamily Proteins (KIFs) as a Fundamental Component of Life: Intracellular Transport and Beyond) and dyneins (Dyneins). An important property of the cytoskeletal components is their ability to form complex structures, which perform elaborate cellular functions. The article Centrioles and the Centrosome discusses centrioles and centrosomes, which are responsible for microtubule organization. The article Cilia and Flagella describes microtubule-based protrusions, cilia and flagella, which are required for motility or sensory and signaling functions. The intricate cytoskeletal machine that drives chromosome separation during cell division, the mitotic spindle, is discussed in article The Mitotic Spindle. The motile and contractile properties of cells and whole organisms strongly depend on actin-based structures, such as filopodia and lamellipodia (Filopodia and Lamellipodia) or muscle fibers (Skeletal Muscle). Formation of complex cytoskeletal assemblies, which is strongly regulated by signaling factors, such as Rho GTPases (Rho GTPases), enables cells to adhere to the extracellular matrix (The Extracellular Matrix; Cell Adhesion to the Extracellular Matrix) or to each other (Cell–Cell Adhesion and the Cytoskeleton), polarize (Cell Polarity), and migrate (Cell Migration).
Intracellular Infectiology The interaction between viral, bacterial, and protozoan pathogens and eukaryotic cells has been of interest to biologists and physicians for over two centuries, since Metchnikoff noted the internalization of bacteria by wandering mobile cells. With the advent of modern cell biology and the emergence of imaging and molecular biology technologies in the past three decades, the field of host defense against pathogens and its failure – resulting in intracellular infection – has advanced at a very rapid pace. Understanding how pathogens access the portals of entry normally reserved for nutrition and defense has intrigued a generation of cell biologists not only from the point of view of expanding our knowledge of normal cell behavior but also of getting a better handle on the molecular mechanisms that invasive organisms use to access intracellular niches where they proliferate. Moreover, understanding the molecular mechanisms of intracellular infection opens up a window of opportunity to develop therapeutics. This section of the Encyclopedia initiates a journey through the various pathways and processes that cells use to kill pathogens by laying out the mechanisms of phagocytosis (Phagocytosis), including a description of the various traffic events that bring the internalized microbes into compartments designed to kill and dismember the invader. Many small particles (and fluid) access the cell by macropinocytosis, a process first described by Lewis almost century ago. In article Macropinocytosis Swanson and Yoshida describe the role of macropinocytosis in cell growth, antigen processing, and infection by virus and
bacteria. Intracellular killing is mediated by reactive oxygen species carried out by complex oxidases, assisted by powerful cationic antimicrobial peptides. Allen (Microbicidal Mechanisms) lays out the basis for intracellular killing as an important component of host defense. Pathogenic bacteria and protozoa have evolved mechanisms to resist killing by host cells; the molecular details of these mechanisms are truly intriguing. Using examples from common pathogens such as Brucella, Chlamydia, Legionella, Listeria, Salmonella, and Shigella, Isberg and colleagues (Bacterial Subversion of Phagocytic Killing) lay out the pathways that intracellular pathogens use to evade detection and killing, thereby permitting residence in intracellular niches where they proliferate. Ferrari and Sansonetti (Cellular Invasion by Bacterial Pathogens) extend this analysis, focusing on the molecular mechanisms of pathogen uptake by nonphagocytic cells and how certain pathogens survive by subverting cellular functions. Lastly, Rigoni and Montecucco (Bacterial Protein Toxins as Tools in Cell Biology and Physiology) discuss potent toxins that increase the possibility of bacterial survival in the face of an effective microbicidal immune system, and how these toxins have become useful reagents in cell biology research. In addition to pathological interactions, the interaction between bacteria and host cells may operate in a saprophytic relationship. Together with the advent of NGS, this concept has opened up a new field called the microbiome (The Gut Microbiome). A second large group of pathogens are the viruses, which need to infect host cells to replicate themselves. Helenius (Cell Biology of Virus Infection) describes the process by which viruses enter cells to exploit the cell’s transport and synthetic pathways. Extending this analysis, Wileman and colleagues (Virus Factories and Mini-Organelles Generated for Virus Replication) describe how various viruses develop factories for efficient intracellular replication. One virus- HIVhas received intense scrutiny at the molecular, cellular, and biomedical/epidemiological levels leading to an impressive while ever improving understanding of its life cycle. Giese, Pelchen-Matthews and Marsh (HIV – The Cell Biology of Virus Infection and Replication) provide a article outlining in detail the Cell Biology of Virus Infection and replication. Over the last two decades another type of disease transmission has emerged, following the discovery of prions. Legname and Pischke (Prions) discuss the cell biology and transmission of scrapie and other prion diseases. Lastly, model organisms being used to understand the relationship between pathogen and host are discussed. Sun and colleagues (Cellular Responses to Infections in Caenorhabditis elegans) describe the cellular responses to infection, using Caenorhabditis elegans as a model organism.
Reference Wolpert, L., 1999. From egg to adult to larva. Evolution & Development 1, 3–4. Available at: https://10.1046/j.1525-142x.1999.00111.x (accessed 05.06.15).
The cell is the building block of life and, as written by Lewis Wolpert (1999), can be considered as the evolution’s most magnificent achievement. Cells, in particular animal cells (plants cells being surrounded by a wall that imposes a constraint), display all kinds of shapes and sizes, and many different types are present in the same living organism (for instance, more than 350 types of cells have been described in humans). Besides this huge diversity, all cells share the same fundamental feature, i.e., the capacity to behave as stable functional units. Such remarkable functional stability allows them to resist change over time and to respond to threats. The cell functional stability relies on its organization in compartments or organelles, which are themselves organized in cellular space by the cell cytoskeleton. Still this organization is not static and the development of live cell imaging approaches in the last 20 years has illustrated the extraordinary dynamics of cell compartments and the multiple connections that exist among them. On the other hand, the loss of stability at the cell level leads to a pathological state, a remarkable example being cancer progression. A loss of stability can also be provoked by pathogens and toxins, which in many cases, can subvert cellular functions to their own benefit. Part II of the Encyclopedia is divided into five sections that describe the range of imaging approaches developed to date to understand the cell organization (see Section Imaging the Cell), the principle organelles (see Section Organelles), the communications between organelles (see Section Interorganellar Communication), how cell architecture is maintained (see Section Cytoskeleton and Motors), and how cell organization can be altered by pathogens (see Section Intracellular Infectiology).
Imaging the Cell The potential of optical methods to acquire a dynamic view of molecules, cells, tissues, and organisms is extraordinary indeed, microscopy is the Dean of Cell Biology. Both light and electron microscopy have moved cell biology into a new era. Yet subcellular structures and organelles, their specific morphological and compositional characteristics, their intimate details and possible connections can be seen only by high-resolution transmission and scanning electron microscopy (TEM and SEM, respectively). TEM has been used for more than 70 years to investigate the ultrastructure of cells and tissues in normal and pathological situations. In more recent times methods have been developed that allow for the analysis of molecular complexes, fine details and 3D analysis of cells,
Encyclopedia of Cell Biology, Volume 2
tissues, small organisms, and subcellular structures including the localization of cellular components with an intraorganellar context. Single-particle cryoEM allows for a near-atomic (B3– 4 Å) resolution of structures or macromolecular complexes such as virus particles, molecular machineries, and membrane proteins. Importantly, these can be viewed in close to native state, without contrasting agents and after vitrification by Cryo-EM (Single-Particle CryoEM of Macromolecular Complexes). Scanning EM has a lower resolution than TEM but it is the only method that permits one to delineate the surface of cells and small organisms and to appreciate structural details thanks to its depth of field and three-dimensional (3D) surface imaging characteristics (Scanning Electron Microscopy in Cell Biology). These classical SEM methods are powerful and have been improved lately by developments in electron microscope technology permitting visualization of isolated organelles and large complexes such as nuclear pores (Scanning Electron Microscopy in Cell Biology). Unfortunately, they do not provide 3D information on the internal characteristics of cells, tissues, and organisms. Over the past few years this limitation has been surpassed by combining SEM imaging with the use of a microtome or a focused ion beam (FIB) that progressively removes sections from the top of the object under study thus uncovering fine details of the interior structures. The acquired images can be stacked and a 3D model can be generated to appreciate the internal organization of a specimen that may be composed of several cells (Imaging Cellular Architecture with 3D SEM). In a very complementary manner and with a higher resolution, Electron Tomography allows one to resolve the 3D organization of molecular and subcellular structures by TEM of plastic embedded sections of cells and tissues and CryoTEM of unstained, vitrified specimens (Electron Tomography). Visualization of molecules within their intracellular and intraorganellar contexts can be achieved using electron dense probes that can be viewed by TEM. Localization methods are most generally based on the recognition of molecules by specific antibodies (Immuno-EM). Such methods combine localization and preservation of ultrastructure and permit discrimination of seemingly identical compartments by their molecular composition in a quantitative manner (Immunoelectron Microscopy: High-Resolution Immunocytochemistry). For many years fluorescence has been the dominant optical property used to study structural and functional properties of cells. Its advantageous qualities include chemical selectivity, i.e., the ability to measure specifically labeled molecular species at very low concentration in the presence of high concentrations of unlabeled molecules, relative ease and speed of measurement, high spatial resolution when used with a
doi:10.1016/B978-0-12-394447-4.20103-X
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Organizational Cell Biology: An Overview
confocal or 2-photon fluorescence microscope and, when used judiciously, lack of interference with normal cell function. The articles in this section of the Encyclopedia provide an introduction to methods that exploit recent advances in microscopy and labeling technology that have opened a new golden age of applications of fluorescence to cell biology. Several of the articles describe methods that enable imaging resolution beyond the Abbe/Rayleigh limit in the range of 200–300 nm for visible light. The oldest of these is total internal reflection microscopy (TIRFM) that provides sub-diffraction optical resolution along the optical axis and so has been a mainstay of studies of dynamic processes of single molecules bound to glass substrates. TIRFM does not, however, improve lateral resolution (Total Internal Reflection Fluorescence Microscopy). Localization methods such as PALM, STORM, and FPALM assemble high-resolution images of macromolecular structures by summing the contributions of component fluorophores whose individual positions are determined with accuracies near 10 nm (Super Resolution Fluorescence Localization Microscopy). As described in article Super-Resolution Light Microscopy: Stimulated Emission Depletion and Ground-State Depletion, STED microscopy is scanning superresolution approach based on restricting the radius of the excitation light to sub-diffraction dimensions. STED has the advantage of acquiring fluorescence signals rapidly enough to be useful in studies of dynamic molecular behavior, for example, via fluorescence correlation spectroscopy (FCS). Structured illumination microscopy (SIM) breaks the diffraction limit by imposing a periodic spatial pattern on the excitation light. Although SIM does not match the resolution of STED and localization methods, it has other advantages of flexibility, relative ease of use and high throughput (Structured Illumination Microscopy). At a higher structural level intravital microscopy (Intravital Microscopy in Mammalian Organisms: From Tissue Physiology to Cell Biology) is used to investigate tissue and cell architecture in living animals and cells. This method has recently benefitted from applications of molecular genetics and improvements in microscopy and fluorescent probes. Fluorescence lifetimes are sensitive to the environment of the emitting fluorophore. Therefore, fluorescence lifetime imaging microscopy (FLIM) provides a unique and popular method for enhancing the contrast of fluorescence images to reveal regional differences in a variety of cellular properties and functions, for example, metabolism, ionic concentrations, and molecular composition (Fluorescence Lifetime Imaging – Applications and Instrumental Principles). Optogenetics (Optogenetics) uses light to trigger molecular functions, and thereby to control activities in living cells. This powerful new approach has mainly been used to control cellular excitability through regulating ion channel function. Although FCS was invented in the 1970s, many variations and extensions have been developed, since then continue to be valuable tools to investigate dynamic molecular processes in cells. The family of FCS and closely related photobleaching (FRAP (fluorescence recovery after photobleaching)) methods yield important information about molecular transport as well as rates and extents of molecular interactions (Fluorescence Correlation Spectroscopy: A Tool for Measuring Dynamic and Equilibrium Properties of Molecules in Cells). While FCS and FRAP rely on dynamic changes in the numbers of fluorescent molecules
detected within a small measurement area that can be no smaller than the optical resolution limit to measure transport rates, single-particle tracking extended by current localization microscopy methods allows measurement of the trajectories of individual molecules with precision in the range of 10 s of nm (High-Speed Localization Microscopy and Single-Particle Tracking). This approach allows a unique analysis of the mechanisms of motion of the tracked molecules. Finally, the use of genetically encoded fluorescent probes coupled with live cell imaging opens a window of opportunity to view cellular events in spectacular detail (Genetically Encoded Fluorescent Probes and Live Cell Imaging).
Organelles Acquisition of diverse intracellular compartments was truly revolutionary epoch in the long history of cellular evolution. Each compartment, known as an organelle, is enclosed by membrane, meaning that cells have a vast area of membrane surface at their disposal, in addition to the plasma membrane surrounding cells, which can act as a platform for biochemical reactions that sustain cellular activities. In addition, the interior space of organelles (the luminal space) sequestered by their limiting membranes also enables cells to perform distinct reactions efficiently. Indeed, organelles contain their own characteristic set of proteins and other molecules to achieve functions unique to each. Study of organelles is undoubtedly one of major streams in cell biology, which started when Robert Brown observed a small dark structure inside plant cells and named it ‘nucleus’ in 1831. This seminal discovery was followed by identification (or naming) of mitochondria by C. Benda and the Golgi apparatus by C. Golgi in the same year 1898, and lysosomes by C. de Duve in 1955. An enormous amount of research from these times has revealed the amazing ‘true colors’ of organelles, but we have not yet completely understood their function and structure; study is still ongoing. This section of the encyclopedia comprehensively covers major organelles including recently discovered and cell-type-specific organelles. Organization of the nucleus, a center for genome storage and for controlling its expression, is introduced in the article Nuclear Organization (Nuclear Structure and Dynamics). There is macromolecular traffic between nucleus and the cytoplasm via the nuclear pore, which is large protein complex inserted into the nuclear double membrane, as discussed in the article Nuclear Pores. Van Anken and Sitia (The Endoplasmic Reticulum) describe the multifunctions of the endoplasmic reticulum; synthesis of lipids and proteins, Caþþ ion storage, signal integration, and interaction with other organelles. Otera and Mihara (Mechanisms and Functions of Mitochondrial Dynamics) focus on mitochondria dynamics; these cellular power plants are highly dynamic and fusion/fission of their membranes play a pivotal physiological role. Cargo internalized into cells from outside is transferred sequentially along the endocytic pathway consisting of early endosomes (Early Endosomal Compartments; Endocytosis of Cargo Proteins: LDL), late endosomes (The Late Endosome), and lysosomes (Conventional and Secretory Lysosomes). Lysosomes are final destination in the pathway, but can fuse with plasma membrane under certain
Organizational Cell Biology: An Overview
conditions (secretory lysosomes), as discussed in the article Conventional and Secretory Lysosomes. In addition, endosomes also function as a platform of signaling, as described in the article Signaling from Endosomes. Membrane traffic from the cytoplasm to lysosomes also exists (the autophagic pathway), which is mediated by autophagosomes (At the Center of Autophagy: Autophagosomes). On the other hand, lipids and proteins synthesized in the endoplasmic reticulum are transported to the Golgi (Golgi and TGN) via intermediate compartments (Intermediate Compartment: A Sorting Station between the Endoplasmic Reticulum and the Golgi Apparatus) to be sorted into each destination. Peroxisomes functioning in oxygen metabolism and fatty acid degradation (Peroxisomes) and lipid droplets specialized for lipid storage and metabolism (Lipid Droplets) are also discussed. Wauben (Extracellular Vesicles) discusses the most-recently discovered organelles, extracellular vesicles, which are released from cells and have diverse important physiological functions. While the abovementioned organelles are ubiquitous in most cell types, there are some cell-type-specific organelles. Only plants and algae have chloroplasts for photosynthesis (Plastids: The Anabolic Factories of Plant Cells). Nerve cells in animals possess synaptic vesicles for neurotransmission, as described in the article Synaptosomes and Synaptic Vesicles. Lastly, Fukuda (LysosomeRelated Organelles) discuss a group of organelles having very specialized functions, for example, pigmentation derived from lysosomes. These lysosome-related organelles exist in various highly differentiated cells.
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multiple pathways from the trans-Golgi network to the cell surface and constitutive and regulated post-Golgi transport are discussed in the articles Post-Golgi Transport – Cargo, Carriers, and Pathways and Regulated versus Constitutive Secretion – A Major Form of Intercellular Communication. Endocytosis is also an important element of interorganellar transport, and this section discusses both the clathrin-mediated and non-clathrinmediated pathways in the articles Clathrin and ClathrinDependent Endocytosis and Clathrin Independent Endocytosis. Transport along the endosomal–lysosomal pathway is covered in the article Endosome to Lysosome Transport, and pathways that carry cargo along the retrograde transport route from the endosomes to the Golgi are discussed in the article Retrograde Transport. Machinery components of vesicle formation and vesicle fusion are essential for the regulation of these transport pathways, and in particular include small G proteins (Rabs of the Endosomal Recycling Pathway; Rabs and Other G Proteins), phosphoinositides (Interorganeller Communication: Role of Phosphoinositides in Membrane Traffic), Bar domain proteins (BAR Domains and BAR Domain Superfamily Proteins), SNAREs (SNAREs: Membrane Fusion and Beyond), ESCRTs (ESCRTing around the Cell), exocyst and tethering factors (Vesicle Tethers) and components of the vesicle coats that interact with cargo, such as adaptor complexes (Adaptor Proteins: Inter-Organelle Traffic Controllers) and retromer (The Retromer Complex). And finally also included are pathways of cell secretion that are independent of the pathway involving the ER and the Golgi, referred to as unconventional protein secretion (Unconventional Protein Secretion: Fibroblast Growth Factor 2 and Interleukin-1β as Examples).
Interorganellar Communication The compartmental organization of the eukaryotic cell is essential for the development of multicellular organisms, for the organization of tissues and organs, and the coordination of physiological functions. The molecular processes which regulate the biogenesis of membrane compartments and the mechanisms by which information is shuttled back and forth between the different intracellular compartments represents fundamental processes in all eukaryotic cells. The pioneering work by Palade and colleagues, which integrated EM technologies with biochemical approaches, laid the foundation for understanding organelle dynamics and interorganellar communication. Subsequent work over the past 3–4 decades has revealed many of the molecular mechanisms which regulate these processes. This section focuses on the molecular mechanisms underlying membrane transport pathways. ER export and ER stress responses are now well-understood and discussed in the articles ER–Golgi Transport and Endoplasmic Reticulum Stress in Disease. Golgi organization and transport is a complex field that has invoked a variety of ingenious approaches in an attempt to define the underlying mechanisms of intracellular transport, as discussed in the article Intra-Golgi Transport. The Golgi is also the site for the glycosylation, and the biosynthesis and role of N-linked glycans and O-glycans in cell biology and development that are discussed in the articles N-Linked Glycans (N-Glycans) and Extracellular O-Glycans, respectively. An emerging field arises from the appreciation that signaling controls transport along the constitutive secretion pathway (Regulation of the Secretory Pathway). Post-Golgi transport involves
Cytoskeleton and Motors The ability of cells to regulate their shape, organize their components, move, and divide depends on the networks of filaments collectively called the cytoskeleton. In contrast to the human skeleton, which remodels slowly, most cytoskeletal filaments are dynamic, because they are composed of polymers of small building blocks that can rapidly assemble and disassemble, depending on the cellular needs. Growth and depolymerization of cytoskeletal filaments can generate forces that can drive cell protrusion and promote chromosome separation during cell division. Moreover, some cytoskeletal filaments can serve as rails for the movement of motor proteins, which use the energy released by ATP hydrolysis to perform mechanical work. The activity of such motors is responsible for the locomotion of single cells, cell contraction and intracellular transport of membrane organelles, RNA, and protein complexes. This section describes the composition, structure, and dynamics of the major eukaryotic cytoskeletal filaments: actin (Actin Assembly Dynamics and Its Regulation in Motile and Morphogenetic Processes), microtubules (Microtubules and Microtubule-Associated Proteins (MAPs)), intermediate filaments (Intermediate Filaments), and septins (Septins: Cytoskeletal Filaments with Structural and Regulatory Functions). A separate article is dedicated to the structurally and functionally diverse cytoskeletal networks present in bacteria and archaea (Bacterial and Archaeal Cytoskeletons). Subsequent articles discuss the molecular mechanisms, motile properties,
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Organizational Cell Biology: An Overview
and functions of the major cytoskeletal motor families. These include the actin-based motors, myosins (Myosins), and the microtubule-based motors, kinesins (Kinesin Superfamily Proteins (KIFs) as a Fundamental Component of Life: Intracellular Transport and Beyond) and dyneins (Dyneins). An important property of the cytoskeletal components is their ability to form complex structures, which perform elaborate cellular functions. The article Centrioles and the Centrosome discusses centrioles and centrosomes, which are responsible for microtubule organization. The article Cilia and Flagella describes microtubule-based protrusions, cilia and flagella, which are required for motility or sensory and signaling functions. The intricate cytoskeletal machine that drives chromosome separation during cell division, the mitotic spindle, is discussed in article The Mitotic Spindle. The motile and contractile properties of cells and whole organisms strongly depend on actin-based structures, such as filopodia and lamellipodia (Filopodia and Lamellipodia) or muscle fibers (Skeletal Muscle). Formation of complex cytoskeletal assemblies, which is strongly regulated by signaling factors, such as Rho GTPases (Rho GTPases), enables cells to adhere to the extracellular matrix (The Extracellular Matrix; Cell Adhesion to the Extracellular Matrix) or to each other (Cell–Cell Adhesion and the Cytoskeleton), polarize (Cell Polarity), and migrate (Cell Migration).
Intracellular Infectiology The interaction between viral, bacterial, and protozoan pathogens and eukaryotic cells has been of interest to biologists and physicians for over two centuries, since Metchnikoff noted the internalization of bacteria by wandering mobile cells. With the advent of modern cell biology and the emergence of imaging and molecular biology technologies in the past three decades, the field of host defense against pathogens and its failure – resulting in intracellular infection – has advanced at a very rapid pace. Understanding how pathogens access the portals of entry normally reserved for nutrition and defense has intrigued a generation of cell biologists not only from the point of view of expanding our knowledge of normal cell behavior but also of getting a better handle on the molecular mechanisms that invasive organisms use to access intracellular niches where they proliferate. Moreover, understanding the molecular mechanisms of intracellular infection opens up a window of opportunity to develop therapeutics. This section of the Encyclopedia initiates a journey through the various pathways and processes that cells use to kill pathogens by laying out the mechanisms of phagocytosis (Phagocytosis), including a description of the various traffic events that bring the internalized microbes into compartments designed to kill and dismember the invader. Many small particles (and fluid) access the cell by macropinocytosis, a process first described by Lewis almost century ago. In article Macropinocytosis Swanson and Yoshida describe the role of macropinocytosis in cell growth, antigen processing, and infection by virus and
bacteria. Intracellular killing is mediated by reactive oxygen species carried out by complex oxidases, assisted by powerful cationic antimicrobial peptides. Allen (Microbicidal Mechanisms) lays out the basis for intracellular killing as an important component of host defense. Pathogenic bacteria and protozoa have evolved mechanisms to resist killing by host cells; the molecular details of these mechanisms are truly intriguing. Using examples from common pathogens such as Brucella, Chlamydia, Legionella, Listeria, Salmonella, and Shigella, Isberg and colleagues (Bacterial Subversion of Phagocytic Killing) lay out the pathways that intracellular pathogens use to evade detection and killing, thereby permitting residence in intracellular niches where they proliferate. Ferrari and Sansonetti (Cellular Invasion by Bacterial Pathogens) extend this analysis, focusing on the molecular mechanisms of pathogen uptake by nonphagocytic cells and how certain pathogens survive by subverting cellular functions. Lastly, Rigoni and Montecucco (Bacterial Protein Toxins as Tools in Cell Biology and Physiology) discuss potent toxins that increase the possibility of bacterial survival in the face of an effective microbicidal immune system, and how these toxins have become useful reagents in cell biology research. In addition to pathological interactions, the interaction between bacteria and host cells may operate in a saprophytic relationship. Together with the advent of NGS, this concept has opened up a new field called the microbiome (The Gut Microbiome). A second large group of pathogens are the viruses, which need to infect host cells to replicate themselves. Helenius (Cell Biology of Virus Infection) describes the process by which viruses enter cells to exploit the cell’s transport and synthetic pathways. Extending this analysis, Wileman and colleagues (Virus Factories and Mini-Organelles Generated for Virus Replication) describe how various viruses develop factories for efficient intracellular replication. One virus- HIVhas received intense scrutiny at the molecular, cellular, and biomedical/epidemiological levels leading to an impressive while ever improving understanding of its life cycle. Giese, Pelchen-Matthews and Marsh (HIV – The Cell Biology of Virus Infection and Replication) provide a article outlining in detail the Cell Biology of Virus Infection and replication. Over the last two decades another type of disease transmission has emerged, following the discovery of prions. Legname and Pischke (Prions) discuss the cell biology and transmission of scrapie and other prion diseases. Lastly, model organisms being used to understand the relationship between pathogen and host are discussed. Sun and colleagues (Cellular Responses to Infections in Caenorhabditis elegans) describe the cellular responses to infection, using Caenorhabditis elegans as a model organism.
Reference Wolpert, L., 1999. From egg to adult to larva. Evolution & Development 1, 3–4. Available at: https://10.1046/j.1525-142x.1999.00111.x (accessed 05.06.15).