Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation.

Rodriguez-Paris, Juan; Nolta, Kathleen V.; Steck, Theodore L.

doi:10.1016/s0021-9258(18)52984-7

Cited by 87 publications

(21 citation statements)

References 43 publications

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“…The results for Ax3 cells (as well as JH10 and HC6) closely resembled those reported by others (1,22,32,41). Specifically, the accumulation of FITC-dextran in Ax3 cells increased in an essentially linear fashion over the first ~60 min, began to plateau by ~90 min, and reached a clear plateau by ~120 min.…”

Section: Single Double and Triple Mutants Exhibit Progressively Greater Decreases In The Rate Of Uptake Of A Fluid Phase Pinocytic Markersupporting

confidence: 87%

“…Specifically, the accumulation of FITC-dextran in Ax3 cells increased in an essentially linear fashion over the first ~60 min, began to plateau by ~90 min, and reached a clear plateau by ~120 min. Others have shown conclu- sively that this plateau is due to the achievement of a steady state wherein the rates of marker uptake and efflux from the cell become equal (22,32,41). The rate of accumulation of the marker, which is always the balance be-tween the rate of uptake and the rate of efflux, does indeed provide the true rate of uptake for times less than 60 rain because there is no appreciable efflux component over at least the first ~6 0 min (unlike vertebrate cells, Dic- the mutants derived from it (A), as well as for representative myoC-(B) and myoD-(C) single mutants, together with their parental cell lines.…”

Section: Single Double and Triple Mutants Exhibit Progressively Greater Decreases In The Rate Of Uptake Of A Fluid Phase Pinocytic Markermentioning

confidence: 99%

“…The time interval between the start of the incubation without extracellular FITC-dextran (time 0 in the plots) and when the amount of cell-associated marker begins to fall represents the transit time. This is the time required for the dextran to traverse the endocytic pathway before egestion (the level of cell-associated dextran remains constant during this time because there is essentially no rapid recycling component) (22,41). As a measure of the efflux rate, we determined the time interval between when cellassociated dextran begins to decrease and when one half (transit time and efflux).…”

Section: Table III Kinetics Of Fluid Phase Pinocytosis (Uptake)*mentioning

confidence: 99%

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Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.

Jung

Hammer

1996

The Journal of Cell Biology

163

187

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Abstract. Dictyostelium cells that lack the myoB isoform were previously shown to exhibit reduced efficiencies of phagocytosis and chemotactic aggregation ("streaming") and to crawl at about half the speed of wild-type cells. Of the four other Dictyostelium myosin I isoforms identified to date, myoC and myoD are the most similar to myoB in terms of tail domain sequence. Furthermore, we show here that myoC, like myoB and myoD, is concentrated in actin-rich cortical regions like the leading edge of migrating cells. To look for evidence of functional overlap between these isoforms, we analyzed myoB, myoC, and myoD single mutants, myoB/myoD double mutants, and myoB/myoC/myoD triple mutants, which were created using a combination of gene targeting techniques and constitutive expression of antisense RNA. With regard to the speed of locomoting, aggregation-stage ceils, of the three single mutants, only the myoB mutant was significantly slower. Moreover, double and triple mutants were only slightly slower than the myoB single mutant. Consistent with this, the protein level of myoB alone rises dramatically during early development, suggesting that a special demand is placed on this one isoform when cells become highly motile. We also found, however, that the absolute amount of myoB protein in aggregation-stage cells is much higher than that for myoC and myoD, suggesting that what appears to be a case of nonoverlapping function could be the result of large differences in the amounts of functionally overlapping isoforms. Streaming assays also suggest that myoC plays a significant role in some aspect of motility other than cell speed. With regard to phagocytosis, both myoB and myoC single mutants exhibited significant reductions in initial rate, suggesting that these two isoforms perform nonredundant roles in supporting the phagocytic process. In triple mutants these defects were not additive, however. Finally, because double and triple mutants exhibited significant and progressive decreases in doubling times, we also measured the kinetics of fluid phase endocytic flux (uptake, transit time, efflux). Not only do all three isoforms contribute to this process, but their contributions are synergistic. While these results, when taken together, refute the simple notion that these three "classic" myosin I isoforms perform exclusively identical functions, they do reveal that all three share in supporting at least one cellular process (endocytosis), and they identify several other processes (motility, streaming, and phagocytosis) that are supported to a significant extent by either individual isoforms or various combinations of them.T o date, 11 classes of myosin have been identified (13,38,47), 10 of which are considered to be unconventional (for review see 12 and 28). While all of these unconventional myosins contain the ~80-kD motor domain that corresponds in sequence to subfragment 1, their nonmotor domain sequences are widely divergent. These unique sequences are thought to confer functional specificity on a more or less gene...

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Section: Single Double and Triple Mutants Exhibit Progressively Greater Decreases In The Rate Of Uptake Of A Fluid Phase Pinocytic Markersupporting

confidence: 87%

Section: Single Double and Triple Mutants Exhibit Progressively Greater Decreases In The Rate Of Uptake Of A Fluid Phase Pinocytic Markermentioning

confidence: 99%

Section: Table III Kinetics Of Fluid Phase Pinocytosis (Uptake)*mentioning

confidence: 99%

See 1 more Smart Citation

Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.

Jung

Hammer

1996

The Journal of Cell Biology

163

187

View full text Add to dashboard Cite

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“…Four such candidates have recently been discussed: the multidrug resistance P-glycoprotein (28), the MTP multidrug resistance protein (29), the NPC1 protein (5), and an unusual anionic phospholipid, called bis(monoacylglycero)phosphate or lysobisphosphatidic acid (30). It is notable that the last three of these are located in late endosomes, lysosomes, and/or lamellar bodies (29)(30)(31)(32)(33), as these are the compartments in which the excess cholesterol accumulates in amphiphile-treated cells (11,27,30,34,35). A block in the export of cholesterol from multivesicular late endosomes could lead to their conversion to lamellar bodies, a related membrane lipid-filled buoyant vacuole (36).…”

Section: Discussionmentioning

confidence: 99%

Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol

Lange

Rigney

et al. 1999

Journal of Lipid Research

219

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The abundance of cell cholesterol is governed by multiple regulatory proteins in the endoplasmic reticulum (ER) which, in turn, are under the control of the cholesterol in that organelle. But how does ER cholesterol reflect cell (mostly plasma membrane) cholesterol? We have systematically quantitated this relationship for the first time. We found that ER cholesterol in resting human fibroblasts comprised ϳ 0.5% of the cell total. The ER pool rose by more than 10-fold in less than 1 h as cell cholesterol was increased by ϳ 50% from below to above its physiological value. The curve describing the dependence of ER on plasma membrane cholesterol had a J shape. Its vertex was at the ambient level of cell cholesterol and thus could correspond to a threshold. A variety of class 2 amphiphiles (e.g., U18666A) rapidly reduced ER cholesterol but caused only minor alterations in the J-curve. In contrast, brief exposure of cells to the oxysterol, 25-hydroxycholesterol, elevated and linearized the J-curve, increasing ER cholesterol at all values of cell cholesterol. This finding can explain the rapid action of oxysterols on cholesterol homeostasis. Other functions have also been observed to depend acutely on the level of plasma membrane cholesterol near its physiological level, perhaps reflecting a cholesterol-dependent structural or organizational transition in the bilayer. Such a physical transition could serve as a set-point above which excess plasma membrane cholesterol is transported to the ER where it would signal regulatory proteins to down-regulate its further accumulation. -Lange, Y., J. Ye, M. Rigney, and T. L. Steck. Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol.

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“…These vacuoles termed postlysosomes are greater than 2.5 m in diameter, contain less vacuolar proton pumps and have lumens that are much less acidic than lysosomes (Nolta et al, 1995). Postlysosomes may arise from fusion of lysosomes (Rodriguez-Paris et al, 1993). Mammalian cells do not contain a compartment analogous to postlysosomes although this type of vacuole may exist in other free living amoebae including the human pathogen Entamoeba histolytica (Aley et al, 1984).…”

Section: ⌬Ddpik1/ddpik2 Cells Are Defective In Transport Of Materials From Lysosomes To Postlysosomes But Are Normal In Transport Proteolmentioning

confidence: 99%

Inactivation of Two Dictyostelium discoideum Genes, DdPIK1 and DdPIK2, Encoding Proteins Related to Mammalian Phosphatidylinositide 3-kinases, Results in Defects in Endocytosis, Lysosome to Postlysosome Transport, and Actin Cytoskeleton Organization

Buczynski

Grove

Nomura

et al. 1997

The Journal of Cell Biology

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Phosphatidylinositide 3-kinases (PI 3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum, DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (Δddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, Δddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme α-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that Δddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1–3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, Δddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

show abstract

Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation.

Cited by 87 publications

References 43 publications

Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.

Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.

Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol

Inactivation of Two Dictyostelium discoideum Genes, DdPIK1 and DdPIK2, Encoding Proteins Related to Mammalian Phosphatidylinositide 3-kinases, Results in Defects in Endocytosis, Lysosome to Postlysosome Transport, and Actin Cytoskeleton Organization

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