Conservation and innovation in Tetrahymena membrane traffic: proteins, lipids, and compartments

ALEJANDRO D. NUSBLAT; LYDIA J. BRIGHT; AARON P. TURKEWITZ

Methods in cell biology

Elsevier

Lugar: Amsterdam; Año: 2012; p. 141 - 175

The partitioning of the cytoplasm into functionally distinct compartments called organelles, delimited by lipidic membranes, is a hallmark of eukaryotic cells.  By concentrating enzymes as well as substrates, each organelle is specialized for a set of reactions, for which compartmental conditions can be optimized.  In addition to increasing the efficiency of individual and coupled reactions, compartmental organization also enables eukaryotic cell to simultaneously execute reactions that might be incompatible if pursued in a continuous cytoplasm. Membrane-bound compartments can also serve for dynamic storage, for example of calcium that can be rapidly transported from the lumen of an organelle via trans-membrane channels to the cytoplasm in response to signaling pathways.  Additionally, molecules within the lumen of organelles can be exported to the cell exterior, which occurs upon fusion of the organellar and plasma membranes. Secretion via membrane fusion is the major mechanism of protein secretion from eukaryotic cells, a phenomenon of great physiological significance that allows cells to influence their environments in a multitude of ways, for both unicellular and multicellular organisms. The secretory apparatus consists of an array of morphologically and biochemically complex organelles, whose concerted activity is responsible for first translocating proteins out of the cytoplasm into the lumen of the secretory pathway, sorting those proteins to accommodate different modes of secretion, and subsequently releasing them into the environment. In many cases the proteins are covalently modified during this process. A second complex network of compartments, partially overlapping with the first, exists to receive and sort molecules that are internalized from the cell surface. A major thrust of eukaryotic cell biology in the last 50 years has been to understand organelle activities and organization.  Broadly speaking, a first aim has been to understand the basic mechanisms underlying organelle biogenesis and function, which are thought to be widely conserved among eukaryotes. Numerous insights have come from exploiting powerful approaches in budding yeast, in many cases providing initial identification of key components, or detailed mechanistic information, for steps that are conserved in mammalian cells. Generally, however, the pathways of membrane traffic in mammalian cells are more complex than in yeast, so a second broad aim has been to understand the additional features of specific mammalian cell types.  For example, some polarized mammalian cells can maintain two or more parallel pathways of protein secretion, with each pathway directed toward a distinct cell surface(Weisz and Rodriguez-Boulan, 2009). These two lines of inquiry have also been explored in evolutionary terms.  In particular, mechanisms that are conserved among eukaryotes are inferred to have been present in a shared eukaryotic ancestor, and therefore very ancient(Dacks and Field, 2007). In contrast, pathways and mechanisms that are specifically required for mammalian complexity can be inferred to have arisen as more recent adaptations that are consequently restricted to a subset of modern lineages. When compared to pathways of membrane traffic in mammalian cells or fungi, the pathways in Tetrahymena are still relatively unexplored.  Many historical studies, as reviewed by Frankel(Frankel, 2000), rely chiefly on morphological analysis. Published electron micrographs provide an excellent overview of many aspects of subcellular organization, and offer a strong starting point for molecular mechanistic studies. However, for many compartments there were and remain few if any identified molecular components. Such molecular markers are powerful tools for direct observations of compartments and their dynamics in living cells (e.g., by green fluorescent protein tagging). Secondly, in some cases molecular markers are essential for interpreting the compartments that are visualized. While some membrane structures are unmistakeable, such as the nuclear envelope, many others are highly pleiomorphic in other cells where they have been studied. Structures such as the Golgi, trans-Golgi network, and distinct classes of endosomes adopt different appearances depending on the cell type and cell activity and can therefore be difficult to identify without molecular markers. Fortunately, the resources available from the sequenced Tetrahymena genome should accelerate the development of such markers, in part by facilitating the identification of Tetrahymena homologs for markers established in other systems. The genomic data also facilitates proteomic approaches to identify the components of isolated organelles. Although many details are lacking, we know that T. thermophila maintains a highly complex network of membrane trafficking pathways. For example, there is evidence for at least four distinct pathways of endocytic uptake. Cells form small endocytic vesicles at cortical invaginations called parasomal sacs(Nilsson and Van Deurs, 1983), while much larger vesicles (phagosomes) arise at the base of the oral apparatus(Nilsson, 1979). While it is clear that different mechanisms are involved in endocytosis from parasomal sacs vs the oral apparatus, neither of these pathways has been dissected in detail. A third pathway of endocytosis, which can be inferred from work in Paramecium, is coupled with the exocytosis of dense-core secretory vesicles and facilitates the rapid recovery of vesicle membranes (Hausmann and Allen, 1976). Fourth, endocytic membrane recovery also occurs upon phagosome fusion, at a cortical site called the cytoproct(Allen and Wolf, 1979). Similarly, Tetrahymena secrete proteins by at least 3 different routes: a pathway of rapid constitutive release of newly-synthesized proteins (for which the vesicular carriers have not been identified)(Bowman and Turkewitz, 2001; Madinger et al., 2010); regulated exocytosis from docked mucocysts(Turkewitz, 2004), and; release of hydrolytic enzymes via lysosome exocytosis(Kiy et al., 1993). There is also indirect evidence for cytoplasmic protein release via an exosome-like mechanism(Madinger et al., 2010). This list understates the complexity of the pathways of cell surface delivery, since for example there is also vesicle trafficking from the cytoproct (the site of phagosome exocytosis) to the oral apparatus(Allen and Fok, 1980; Bright et al., 2010). It is also not clear whether there is a single pathway of endocytosis from parasomal sacs or whether parallel pathways exist, as will be discussed below.             In our view, more detailed molecular studies of trafficking in T. thermophila could be significant for several reasons. While it may not be possible or even desirable to obtain a comprehensive understanding of the entire network of trafficking steps, there are individual pathways whose analysis could make major contributions to both of the aims outlined above. Unlike budding yeast whose successful evolutionary strategy was to become small and relatively simple, the ciliates, like animal cells, have undergone large expansions in gene families encoding key determinants of membrane trafficking, as the membrane trafficking pathways themselves grew increasingly complex(Bright et al., 2010; Eisen et al., 2006; Saito-Nakano et al., 2010). Because the adaptations to membrane traffic occurred independently in ciliates and animals, a comparison of these lineages offers one the chance to ask whether specific pathways were prone to expansion and adaptation. This question is also being asked more broadly, taking advantage of the wealth of sequenced genomes now available to ask whether the determinants of specific pathways have tended to expand in multiple lineages, to generate large gene subfamilies. For example, phylogenetic analysis of SNARE proteins in many lineages suggests that SNARE subfamilies associated with endocytosis have undergone more expansion than other subfamilies, suggesting that endocytosis has been a particularly rich substrate for innovations in membrane traffic(Kienle et al., 2009). These metagenomic studies can be complemented by more in-depth studies of individual organisms. Studying such questions in a specific complex non-animal lineage, such as higher plants or ciliates, allows one to confirm the phylogenetic predictions, i.e., test the underlying assumption that sequence comparison are reliable for assigning function. Secondly, such single-species studies are critical to understanding both how, and to what purpose, the genetic innovations have modified conserved pathways or generated new ones. In other words, how has selection acted on the organization and function of membrane trafficking pathways? Critically, the experimental tools available in T. thermophila already facilitate asking complex cell biological questions, and new approaches continue to be developed(Turkewitz et al., 2002). One such novel approach to studying membrane traffic in particular is discussed at the end of this chapter, based on the availability of extensive whole genome expression data. Effective use of T. thermophila may come from exploiting unique features of its complex and unusual organization. For example, many sites of specific membrane trafficking steps at the plasma membrane are organized as precise arrays, allowing a microscopist to analyze multiple sites, simultaneously, at predictable locations. This aspect of ciliate organization has recently been brilliantly exploited to analyze basal bodies(Pearson and Winey, 2009). For membrane trafficking, such organized domains include sites of clathrin-mediated endocytosis and of regulated exocytosis(Allen, 1967; Elde et al., 2005; Satir et al., 1973). A second striking aspect of Tetrahymena is that there are structurally and functionally distinct variants of several organelles, maintained in the same cytoplasm. This is best known for the nucleus, where studies exploiting the differences between the macro- and micronucleus have made pivotal contributions to molecular biology(Pederson, 2010). Nuclear dimorphism in Tetrahymena has recently been exploited to analyze the role of nuclear pore components. Nuclear pores are selective gates that regulate traffic of cytosolic and membrane proteins into the nucleoplasm, and a major question in the field is how the components of nuclear pores act as gatekeepers, with much attention focusing on iterative motifs consisting of glycine-leucine-phenylalanine-glycine (GLFG) that are abundant in many proteins lining the pores (nucleoporins, or nups). In ciliates, the two functionally-distinct nuclei contain different sets of nucleoplasmic proteins, implying that nuclear pores in Mics and Macs are also distinct. Haraguchi and colleagues recently identified micronuclear- and macronuclear-specific versions of NUP98(Iwamoto et al., 2009). The repeats in the micronuclear (but not macronuclear) Nup98p were NIFN, rather than the canonical GLFG, and domain-swapping experiments provided evidence that the change in the Nup repeat motif has functional consequences for gatekeeping. This line of work therefore holds promise both to reveal mechanisms underlying nuclear dimorphism in Tetrahymena as well as providing a unique model system for dissecting features of nuclear pore selectivity. Another example of organellar differentiation in Tetrahymena is that each cell contains both a “standard” endoplasmic reticulum (ER), including the nuclear envelope, but also distinct flattened cisternae called alveoli that tightly underlayer the plasma membraneto. While alveoli have been only glancingly studied in Tetrahymena, data from Paramecium make a strong case that alveoli function as a major store for mobilizable calcium, a classical activity of the ER, and also contain some ER proteins(Plattner et al., 1999; Stelly et al., 1995). ER subdomains in animal cells are recognized as an important aspect of the secretory pathway and of cellular signaling, and understanding the biogenesis and maintenance of the ER and alveoli in Tetrahymena may offer exceptional opportunities for illuminating mechanisms of protein and lipid sublocalization in this organelle. Lastly, Tetrahymena, because of the strong experimental tools that have been developed, may be an excellent organism to appreciate “cell biodiversity”, namely the range of adaptations that have evolved in eukaryotes that are deeply divergent from animals. For example, the contractile vacuole is a multi-part organelle that collects water from the cytoplasm to pump it out of the cell, and is essential for osmotic homeostasis in fresh water organisms lacking cell walls. The remarkable properties of the ciliate contractile vacuole have been investigated in Paramecium, but virtually nothing is known about assembly or mechanism of action at the molecular level(Allen, 2000). Contractile vacuoles are also present in Amoebozoa and other distantly-related lineages, but whether these are homologous organelles to those in Ciliates (i.e., inherited from a common ancestor), or whether organelles as complex as contractile vacuoles have arisen multiple times, independently, is an open question. Pursuing such organelles in Tetrahymena, as they are also being studied in Dictyostelium, could help to provide a new perspective on the relative importance of inheritance vs innovation in the structures that animate modern eukaryotes.