BBA - General Subjects (v.1525, #3)

Editorial by James Cardelli (vii).

The main route for fluid-phase uptake in Dictyostelium is macropinocytosis, a process powered by the actin cytoskeleton. Nutrients within the endocytosed fluid are digested and resorbed, disposal of remnants follows by exocytosis. Along the endocytic pathway, membrane fusion and fission events take place at multiple steps. The regulator and effector molecules involved in uptake and transit are largely conserved between higher and lower eukaryotes. This feature, together with its accessibility by molecular genetics, recommend Dictyostelium as a valuable model system for mammalian cells.
Keywords: Endocytosis; Macropinocytosis; Cytoskeleton; Actin; Lysosome; Dictyostelium;

Phagocytosis, a critically important process employed by leukocytes against invading pathogens, is an actin-dependent clathrin-independent process that results in the internalization of particles >0.5 μm in diameter. Phagocytosis consists of a number of stages, including the binding of particles to the cell surface via interaction with a receptor, engulfment of the particle by pseudopod extension, and fission and fusion reactions to form phago-lysosomes. Much remains to be learned concerning the molecular mechanisms that regulate particle internalization and phagosome maturation. Dictyostelium is a genetically tractable professional phagocyte that has proven useful in determining the molecular steps involved in these processes. We will summarize, in this chapter, what we currently understand concerning the molecular mechanisms that regulate the process of phagocytosis in Dictyostelium, and we will compare and contrast this body of information with that available describing phagocytosis in higher organisms. We will also present current information that suggests that macropinocytosis, a process morphologically similar to phagocytosis, utilizes a different signaling pathway than phagocytosis. Finally, we will discuss the process of maturation of phagosomes, which requires membrane trafficking events, and we will summarize data that support the use of Dictyostelium as a model to determine how intracellular pathogens survive.
Keywords: Macropinocytosis; Phosphatidylinositol 3-kinase; Rab GTPase; Phagosome; Phagocytosis;

The regulation of actin polymerization and cross-linking in Dictyostelium by Eunkyung Lee; Ka-ming Pang; David Knecht (217-227).
It is clear that the polymerization and organization of actin filament networks plays a critical role in numerous cellular processes. Inhibition of actin polymerization by pharmacological agents will completely prevent chemotactic motility, macropinocytosis, endocytosis, and phagocytosis. Recently there has been great progress in understanding the mechanisms that control the assembly and structure of the actin cytoskeleton. Members of the Rho family of GTPases have been identified as major players in the signal transduction pathway leading from a cell surface signal to actin polymerization. The Arp2/3 complex has been added to the list of means by which new actin filaments can be nucleated. However, it is clear that actin polymerization by Arp2/3 complex is not the whole story. In principle, the final structures formed by actin filaments will depend on factors such as: the length of actin filaments, the degree of branching, how they are cross-linked and the tensions imparted on them. In addition, the means by which actin polymerization generates protrusion of membranes is still controversial. A phagosome, filopodium and a lamellipodium all require polymerization of new actin filaments, but each has a characteristic morphology and cytoskeletal structure. In the following chapter, we will discuss actin polymerization and filament organization, especially as it relates to the machinery of phagocytosis in Dictyostelium.
Keywords: Actin; Phagocytosis; GTPase; Cytoskeleton; Actin binding protein; Actin cross-linking protein; Rho; Rac; Signal transduction;

Profilin is a key regulator of actin polymerization, and plays a pivotal role at the interface of the phosphoinositide signal transduction pathway and the cytoskeleton. Recent evidence suggests the involvement of profilin in the regulation of phagocytosis and macropinocytosis, and the transport along the endosomal pathway. Disruption of profilin leads to a complex phenotype that includes abnormal cytokinesis, a block in development and defects in the endosomal pathway. Macropinocytosis, fluid phase efflux and secretion of lysosomal enzymes were reduced, whereas the rate of phagocytosis was increased as compared to wild-type cells. The lmpA gene, a homolog of the CD36/LIMPII family, was identified as a suppressor for most of the profilin-minus defects. This gene encodes an integral membrane protein, it localizes to lysosomes and macropinosomes, and binds to phosphoinositides. Even though phosphatidylinositol lipids constitute only a small fraction of total lipids in the membranes of eukaryotic cells, they play an important role in vesicle transport, signal transduction and cytoskeletal regulation. Disruption of lmpA in wild-type cells resulted in defects in fluid phase efflux and macropinocytosis, but not in phagocytosis. The discovery and initial characterization of two additional members of the CD36/LIMPII family in Dictyostelium, lmpB and lmpC, that exhibit intriguing differences in developmental regulation and their putative sorting signals, suggests that a set of lysosomal integral membrane proteins contribute to the crosstalk between vesicles and cytoskeletal proteins.
Keywords: Dictyostelium; Lysosomal integral membrane proteins; Phagocytosis; Phosphoinositides; Profilin;

Molecular motors and membrane traffic in Dictyostelium by Shuo Ma; Petra Fey; Rex L Chisholm (234-244).
Phagocytosis and membrane traffic in general are largely dependent on the cytoskeleton and their associated molecular motors. The myosin family of motors, especially the unconventional myosins, interact with the actin cortex to facilitate the internalization of external materials during the early steps of phagocytosis. Members of the kinesin and dynein motor families, which mediate transport along microtubules (MTs), facilitate the intracellular processing of the internalized materials and the movement of membrane. Recent studies indicate that some unconventional myosins are also involved in membrane transport, and that the MT- and actin-dependent transport systems might interact with each other. Studies in Dictyostelium have led to the discovery of many motors involved in critical steps of phagocytosis and membrane transport. With the ease of genetic and biochemical approaches, the established functional analysis to test phagocytosis and vesicle transport, and the effort of the Dictyostelium cDNA and Genome Projects, Dictyostelium will continue to be a superb model system to study phagocytosis in particular and cytoskeleton and motors in general.
Keywords: Membrane traffic; Molecular motor; Kinesin; Dynein; Myosin; Actin cytoskeleton; Microtubule;

Regulation of Dictyostelium Myosin I and II by Marc A. de la Roche; Graham P. Côté (245-261).
Dictyostelium expresses 12 different myosins, including seven single-headed myosins I and one conventional two-headed myosin II. In this review we focus on the signaling pathways that regulate Dictyostelium myosin I and myosin II. Activation of myosin I is catalyzed by a Cdc42/Rac-stimulated myosin I heavy chain kinase that is a member of the p21-activated kinase (PAK) family. Evidence that myosin I is linked to the Arp2/3 complex suggests that pathways that regulate myosin I may also influence actin filament assembly. Myosin II activity is stimulated by a cGMP-activated myosin light chain kinase and inhibited by myosin heavy chain kinases (MHCKs) that block bipolar filament assembly. Known MHCKs include MHCK A and MHCK B, which have a novel type of kinase catalytic domain joined to a WD repeat domain, and MHC-protein kinase C (PKC), which contains both diacylglycerol kinase and PKC-related protein kinase catalytic domains. A Dictyostelium PAK (PAKa) acts indirectly to promote myosin II filament formation, suggesting that the MHCKs may be indirectly regulated by Rac GTPases.
Keywords: Myosin I; Myosin II; p21-activated kinase; Myosin II heavy chain kinase; Dictyostelium;

Signalling pathways based on the small GTPase Ras regulate a multitude of cellular events in eukaryotic cells. Dictyostelium expresses a large and varied family of Ras proteins. It also uses a range of known Ras regulators, in particular RasGEFs, and effectors. The genetic tractability of Dictyostelium, together with the wide range of Ras proteins and regulators, make it an ideal model for the genetic dissection of Ras pathways. This review highlights the recent advances in our understanding of Ras function in Dictyostelium, and considers the implications of these findings for our understanding of eukaryotic signal transduction.
Keywords: Small GTPase; Endocytosis; Cell; Motility; RasGEF; Guanine nucleotide exchange factor; Dictyostelium discoideum;

Contents Vol. 1525 (274-275).