Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.

Abstract

Abstract. The functional relationship between three Dictyostelium myosin Is, myoA, myoB, and myoC, has been examined through the creation of double mutants.Two double mutants, myoA-/B-and myoB-/C-, exhibit similar conditional defects in fluid-phase pinocytosis. Double mutants grown in suspension culture are significantly impaired in their ability to take in nutrients from the medium, whereas they are almost indistinguishable from wild-type and single mutant strains when grown on a surface. The double mutants are also found to internalize gp126, a ll6-kD membrane protein, at a slower rate than either the wild-type or single mutant cells. Ultrastructural analysis reveals that both double mutants possess numerous small vesicles, in contrast to the wild-type or myosin I single mutants that exhibit several large, clear vacuoles. The alterations in fluid and membrane internalization in the suspension-grown double mutants, coupled with the altered vesicular profile, suggest that these cells may be compromised during the early stages of pinocytosis, a process that has been proposed to occur via actin-based cytoskeletal rearrangements. Scanning electron microscopy and rhodamine-phalloidin staining indicates that the myosin I double mutants appear to extend a larger number of actin-filled structures, such as filopodia and crowns, than wild-type cells. Rhodamine-phalloidin staining of the F-actin cytoskeleton of these suspension-grown cells also reveals that the double mutant cells are delayed in the rearrangement of cortical actinrich structures upon adhesion to a substrate. We propose that myoA, myoB, and myoC play roles in controlling F-actin filled membrane projections that are required for pinosome internalization in suspension.

Molecular Mechanisms of Membrane Trafficking. What do we Learn from Dictyostelium discoideum?

“…Myosins of the class I have been localised to membrane ruffles and phagocytic cups in D. discoideum (Fukui et al 1989;Morita et al 1996), Entamoeba histolytica (Voigt et al 1999), Acanthamoeba castellanii (Baines et al 1992;Baines et al 1995;Jontes et al 1998) and macrophages (Allen and Aderem 1995;Swanson et al 1999). D. discoideum cells lacking either myosin IB, IC or IK exhibit significant reduction of the initial rate of particle uptake [Jung et al 1996; Schwarz EC, Neuhaus EM, Kistler C, and Soldati T: Dictyostelium MyoK, a novel type of myosin with crucial functions in motility and maintenance of cortical integrity (submitted)], and double deletion mutants (myoA-/myoB-, myoC-/myoB-) show conditional defects in fluid-phase pinocytosis (Jung et al 1996;Novak et al 1995).…”

Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition

“…Myosin-1 is able to cross-link the plasma membrane to the underlying actin cytoskeleton and plays a direct role in the control of cortical and membrane tension (11,12). These motor proteins can also drive vesicle trafficking, pseudopod retraction, and the uptake of particles and fluids by phagocytosis and micropinocytosis (13)(14)(15)(16).…”

Unconventional myosins at the crossroad of signal transduction and cytoskeleton remodeling

“…This domain has been shown to serve as an ATP-independent secondary actin-binding site (56,57). In good agreement with the presence of both membrane-and actin-binding sites, myoB, myoC, and myoD have been localized at the cortex of vegetative cells and at the leading edge of motile D. discoideum cells during chemotaxis (13,(71)(72)(73)(74). TH3 is composed of an SH3 domain which has been suggested to either bind to yet undefined partners or intramolecularly to the Pro-rich TH2 domain.…”

“…Nevertheless, it has been shown that phagocytosis (7) and references therein) as well as part of axonal transport (8) and exocytosis (9) are actin-based movement. Endocytosis in yeast (10), vertebrates (11) and amoebae (12,13), polarized secretion in yeast (14), and fungi (15), vacuole inheritance in yeast (16), contractile vacuole function in amoebae (17), as well as blastoderm cellularization and cytoplasmic transport of particles in Drosophila (18,19), require specific myosin motors. Myosin V has now been shown to power the redistribution of melanosomes in mouse melanocytes (20,21), as well as in fish and Xenopus melanophores (22,23).…”

“…Disruptions of single myosin I genes showed only subtle alterations of cell motility, and multiple disruptions had to be performed to induce stronger phenotypic impairments. Different combinations of double and even triple mutations of myoB, myoC, myoD, or myoA resulted in strains impaired to different extents in macropinocytosis and motility, highlighting the potentially high degree of functional redundancy (12,13,75). It is important to note that in a similar way, disruption of the genes for multiple actin-binding proteins was necessary to reveal strong phe-Exploring the Myosin Repertoire 399 notypic alterations (76), indicating that a certain level of redundancy is probably inherent to components of the actin cytoskeleton and associated vital functions.…”