Plasma Membrane

Many cells of the myeloid lineage use an unusual secretory organelle to deliver their effector mechanisms. In these cells, the lysosomal compartment is often modified not only to fulfill the degradative functions of a lysosome but also as a mechanism for secreting additional proteins that are found in the lysosomes of each specialized cell type. These extra proteins vary from one cell type to another according to the specialized function of the cell. For example, mast cells package histamine; cytotoxic T cells express perforin; azurophilic granules in neutrophils express antimicrobial peptides, and platelets von Willebrand factor. Upon release, these very different proteins can trigger inflammation, cell lysis, microbial death, and clotting, respectively, and hence deliver the very different effector mechanisms of these different myeloid cells.

Strain variability is a central topic in the discussion of Candida biology. Most of the Candida isolates that are studied are derived from clinical specimens. Therefore, a discussion of variability among Candida isolates within a particular species starts with decisions that are made in the diagnostic microbiology laboratory. Studies of Candida genetic variability may also be conducted from the perspective of assessing genomic rearrangements. The nature of genomic rearrangements in the diploid Candida albicans was understood more clearly from construction of a physical map of its eight pairs of chromosomes. DNA fingerprinting probe sequences useful in phylogenetic and epidemiological studies are derived from the major repeat sequence (MRS). The MRS is also present in the closely related Candida dubliniensis. Events such as mutation and mitotic recombination also can contribute to variability among C. albicans strains. One study examined whether the local wildlife population was responsible for maintaining a reservoir of C. albicans isolates specific to a defined geographic area in the midwestern United States. The work was expanded to include collection of C. albicans isolates from domestic animals. Results showed that there is a significant difference in the clade distribution of isolates from humans and wildlife, demonstrating population isolation between the groups. The work demonstrates the impressive display of genetic variability that C. albicans can develop when challenged with exposure to antifungal drugs. A given isolate of C. albicans can undergo an impressive range of genetic changes at the level of point mutation to alterations in whole chromosomes.

Chlamydiae are taken up by host cells so efficiently that the process has been termed "parasite-specified endocytosis" by Byrne and Moulder. This chapter focuses upon the active interactions of chlamydiae with the host cell and covers the stage of development up to the division of reticulate bodies (RBs). Surprisingly, different mutant cell lines displayed unique patterns of susceptibility or resistance to different C. trachomatis serovars or to C. pneumoniae. Despite the difficulties of comparing studies between different chlamydial species or serovars, conditions, and cell types, there is sufficient evidence to suggest that individual chlamydiae are capable of utilizing different mechanisms for entry. Translocation of Tarp and its tyrosine phosphorylation appear to be one of the first means of communication with the host cell to actively subvert host processes for parasite purposes. Identification of the kinases mediating Tarp phosphorylation is an initial step in mapping the signal transduction networks initiated by C. trachomatis to establish residence within an intracellular niche. Endocytosed elementary bodies (EBs) are rapidly transported to the peri-Golgi region of the host cell and become fusogenic with Golgi-derived vesicles. The Inc proteins do not share structural similarities to eukaryotic proteins, except for SNARE-like motifs, and homology searches do not provide substantial clues about their function. An improved understanding of the complex interactions of the important pathogens with the host cell should provide great potential for improved chemotherapeu-tic and immunoprophylactic interventions.

Viruses are the most abundant infectious agents on earth and the most primitive form of life, predating us by billions of years. Due to the fact that viruses utilize host cell machinery to replicate, the host has the daunting task of distinguishing virus infection signatures from self-molecules. This chapter provides an overview of the mechanisms used to sense virus infection (innate recognition), the cytokine system that evolved to rapidly induce antiviral states in neighboring cells (type I interferons), and the methods used to contain and destroy viruses (effector functions). Analysis of animals deficient in the RIG-I-like receptors (RLRs) revealed important and distinct roles for the sensing molecules in innate immunity. DNA viruses such as adenovirus stimulate the NLRP3-ASC-caspase-1 inflammasomes in vivo. The existence of a TLR-independent cytoplasmic DNA sensing molecule leading to type I IFN production was suggested from studies utilizing DNA viruses and bacteria. The mechanism of RNAi involves two steps. First, viral dsRNA is recognized by Dicer-like endonuclease family, which processes it into siRNA. Second, the siRNA are incorporated into RNA-induced silencing complex (RISC), which guide the RNase enzyme AGO to complementary sequences (viral RNA) for cleavage and degradation of viral RNA. Tetherin associates with lipid rafts and inhibits retrovirus particle release in the absence of Vpu. Vpu utilizes the beta-TrCP E3 ubiquitin ligase complex to induce endosomal trafficking events that remove tetherin from the cell surface, rendering it incapable of restricting the release of enveloped viruses.

This chapter summarizes selected and somewhat disparate advances made in yeast cytology after about 1950. Northcote and Horne disintegrated their baker’s yeast mechanically, and after centrifuging, mounted the cell wall fraction in polyvinyl formal films. The yeast walls proved to be stratified: after acid hydrolysis, chromatography showed that the outer layer was mainly mannan-protein; the walls contained 29% glucan and 31% mannan, previously reported for yeast cell walls as 13% protein and 8.5% lipid . It was in 1956 that Necas described the formation of some protoplasts or spheroplasts amongst spontaneously autolyzing S. cerevisiae. In 1989, Hartwell emphasized the relevance of this work on the cell cycle for the prevention of mammalian cancers. Chromosome behaviour in meiosis is well characterized from cytological and genetic descriptions but little is known of the underlying molecular mechanisms, largely because no one experimental system has been developed to support an integrated application of modern cytological, genetic, and molecular biological methods. A thorough account of the amount of work published on yeast cytology in the last 50 or so years would occupy several volumes; in addition, no account has been given here of advances made since 1990, which involve such innovations as the green fluorescent protein reporter system, confocal microscopy, and flow cytometric analysis.

The ability to understand cytoplasmic structure can provide powerful insights into the biology of cells and organisms. This chapter has briefly reviewed the diversity of hyphal structures and presented examples of how bioimaging has contributed to a broader understanding of hyphal biology and phylogenetic relationships between fungal taxa. At the heart of polarized growth is the secretory pathway in which vesicles are targeted to sites of growth and subsequently fuse with the plasma membrane. In mature hyphae of the septate fungi, these events have given rise to the Spitzenkörper, a complex and dynamic structure that clearly influences hyphal growth and morphogenesis. The presence or absence of certain morphological characters (e.g., septa and Woronin bodies) already has been useful in defining higher taxa, especially since evolutionary polarity often can be established using stable phylogenetic trees based on DNA sequences. Ever-enlarging molecular databases, especially those of whole genomes, are allowing us to look for the genetic basis of many structural features, such as the presence or absence of Woronin body matrix proteins. This capability will allow us to understand the basis of these features not only in an evolutionary sense but also in a functional one. Collaboration among different types of fungal biologists including systematists is essential to understanding structure and how it applies to the study of the Fungi.

This chapter focuses on the microtubule and actin cytoskeletons of filamentous fungi, including the motor proteins that are integral to cytoskeletal function. Relevant results from yeasts are discussed to provide background and context. The emphasis is on more recent data from live-cell imaging as well as genetic and molecular genetic studies. It has been shown that both the microtubule and the actin cytoskeletons play roles in polarized growth of hyphae, and how these cytoskeletal elements function to support hyphal growth and organelle distribution in elongated hyphae is a topic of great interest. Dynein in filamentous fungi also participates in organizing the microtubule network by regulating microtubule dynamics and by providing force for transporting microtubules. In filamentous fungi, the actin cytoskeleton and its myosin motors are important for the delivery of cell membrane and cell wall components to the growing hyphal tip and to the septum. Myosins are a diverse superfamily of actin motor proteins that play various cellular roles. In filamentous fungi such as A. nidulans and N. crassa, four families of myosins have been found, including myosin-I, myosin-II, myosin-V, and the fungus-specific chitin synthases with myosin motor domains. Hyphal growth in filamentous fungi needs both microtubule and actin cytoskeletons, and thus, it would be important to understand how these two systems interact to coordinate vesicle transport towards the hyphal tip.

At the turn of the 21st century, researchers linked the second messenger bis-(3',5') cyclic diguanylic acid (also known as cyclic di-GMP [c-di-GMP]) to proteins that contained either the GGDEF domain, the EAL domain, or both and showed that these proteins were ubiquitous. Researchers had reported that epinephrine was produced by the adrenal gland and that this epinephrine traveled from the adrenal gland to the liver cells that store glycogen; however, the mechanism by which epinephrine elicited this effect remained unknown. Today, the mammalian cyclic AMP (cAMP) signal transduction network is known to include a dizzying array of G-protein-coupled receptors, a plethora of G-protein subunits, 10 adenylyl cyclases (9 membrane bound and 1 cytosolic), 11 PDE families, and multiple cAMP effectors, including PKA, PKC, diverse guanine exchange factors, and a variety of cyclic nucleotide-gated ion channels. The parallels between the mammalian cAMP network and the bacterial systems centered on c-di-GMP are striking. One obvious reason to compartmentalize proteins within microdomains is to bring related signaling components into close proximity. The mammalian plasma membrane is heterogeneous. This heterogeneity is produced, in part, by the concentration to specific locations of large amounts of cholesterol and sphingolipids. Two major hypotheses have been proposed to explain the ability of PDEs to shape cAMP gradients: the barrier hypothesis and the sink hypothesis.

Phagocytes ingest a variety of particles, cells, and microbes by use of actin-rich, contractile extensions of plasma membrane that close into intracellular membranous organelles called phagosomes. Signaling for phagocytosis begins when cell surface receptors engage particle-bound molecular ligands. The distinct component activities localize to subregions of forming phagosomes and may be coordinated by membrane-associated proteins and phospholipids that diffuse in the plane of the membrane bilayer. Different receptors use distinct mechanisms for regulating the actin cytoskeleton and the movements of membrane, producing a variety of morphologies for forming phagosomes. Some elements of phagocytic signaling are both constructive and interpretive. This chapter emphasizes the constructive signals. The cytosolic signaling proteins bind to the receptors or associated proteins, and a complex aggregate assembles around the receptor by diffusion and trapping. Secondary signals include the enzymatic modification of phospholipids and other membrane-associated proteins that in turn organize the cytoplasmic movements of phagocytosis. The underlying mechanism of phagocytosis appears to depend less on localized extension of the plasma membrane and more on controlled invagination of the cell surface by actin-myosin contractile activities. The morphogenesis of phagosomes suggests that actinmyosin-like contractility is essential to phagocytosis. The chapter reviews the signals for various different receptor-mediated phagocytic processes. The patterns of GTPase activities and the corresponding patterns of movement for actin, myosin, and membranes may be coordinated by patterns of membrane lipids in the inner leaflet of the inner membrane of the phagocytic cup that appear and disappear through the course of phagocytosis.

Autophagic degradation is a cell-autonomous mechanism for direct elimination of intracellular microbes, but also plays a broader role in innate and adaptive immunity and in general cytoplasmic homeostasis. The process of autophagy plays many functions, including feeding the cells under starvation conditions or upon withdrawal of growth factors. Autophagy plays both housekeeping and immune functions in macrophages and dendritic cells. The phagophore is enlarged by the addition of a new membrane that is of undefined origin but is suspected to come from the endoplasmic reticulum or a combination of sources including Golgi and endosomes. Most cells undergo baseline autophagy, removing protein aggregates and spuriously damaged mitochondria or other organelles, or adjusting the cellular biomass. The classical physiological inducers of autophagy are amino acid starvation or absence of growth factors. Recent studies examining effects on the redistribution of a sole integral membrane Atg protein, Atg9, have implicated ULK1 as the putative mammalian Atg1 ortholog. A classical physiological inducer of autophagy is amino acid starvation, in particular, withdrawal of leucine. In mammalian cells, the role of autophagy has been linked to immunological signals. In mice and humans it is under the control of cytokines and agonists regulating innate and adaptive immunity, such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 13 (IL-13), and CD40L-CD40.