scholarly journals Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants

1996 ◽  
Vol 109 (3) ◽  
pp. 663-673 ◽  
Author(s):  
L.A. Temesvari ◽  
J.M. Bush ◽  
M.D. Peterson ◽  
K.D. Novak ◽  
M.A. Titus ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Dictyostelium Discoideum ◽  
Cell Lines ◽  
Double Mutant ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Contractile Vacuole ◽  
C Cell ◽  
Myosin I ◽  
Lysosomal System

The role of myosin Is in endosomal trafficking and the lysosomal system was investigated in a Dictyostelium discoideum myosin I double mutant myoB-/C-, that has been previously shown to exhibit defects in fluid-phase endocytosis during growth in suspension culture (Novak et al., 1995). Various properties of the endosomal pathway in the myoB-/C- double mutant as well as in the myoB- and myoC- single mutants, including intravesicular pH, and intracellular retention time and exocytosis of a fluid phase marker, were found to be indistinguishable from wild-type parental cells. The intimate connection between the contractile vacuole complex and the endocytic pathway in Dictyostelium, and the localization of a myosin I to the contractile vacuole in Acanthamoeba, led us to also examine the structure and function of this organelle in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in the myoB-, myoC- or myoB-/C- cell lines. The transport, processing, and localization of a lysosomal enzyme, alpha-mannosidase, were also unaltered in all three mutants. However, the myoB- and myoB-/C- cell lines, but not the myoC- cell line, were found to oversecrete the lysosomal enzymes alpha-mannosidase and acid phosphatase, during growth and starvation. None of the mutants oversecreted proteins following the constitutive secretory pathway. Two additional myosin I mutants, myoA- and myoA-/B-, were also found to oversecrete the lysosomally localized enzymes alpha-mannosidase and acid phosphatase. Taken together, these results suggest that these myosins do not play a role in the intracellular movement of vesicles, but that they may participate in controlling events that occur at the actin-rich cortical region of the cell. While no direct evidence has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fidelity of the actin cortex, and changes in cortical organization could influence lysosomal-related membrane events such as internalization or transit of vesicles to the cell surface.

scholarly journals Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

1997 ◽  
Vol 8 (7) ◽  
pp. 1343-1360 ◽  
Author(s):  
G Buczynski ◽  
J Bush ◽  
L Zhang ◽  
J Rodriguez-Paris ◽  
J Cardelli
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Lines ◽  
Lysosomal Enzymes ◽  
Amino Acid Level ◽  
Active Form ◽  
Fluid Phase ◽  
Retrograde Transport ◽  
Dominant Negative ◽  
Lysosomal Hydrolases ◽  
Small Molecular Weight

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexposed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles, the size of postlysosomes (> 2.5 microns) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, > 90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H(+)-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.

scholarly journals A Myosin I Is Involved in Membrane Recycling from Early Endosomes

2000 ◽  
Vol 150 (5) ◽  
pp. 1013-1026 ◽  
Author(s):  
Eva M. Neuhaus ◽  
Thierry Soldati
Keyword(s):  
Plasma Membrane ◽  
Fluid Phase ◽  
Surface Proteins ◽  
Endocytic Pathway ◽  
Membrane Recycling ◽  
Myosin I ◽  
Early Endosomes ◽  
Butanedione Monoxime ◽  
Microscopy Method ◽  
Membrane Components

Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin–myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA−/B−, and myoB− cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA−/B− cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.

scholarly journals A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum.

1996 ◽  
Vol 7 (10) ◽  
pp. 1623-1638 ◽  
Author(s):  
J Bush ◽  
L Temesvari ◽  
J Rodriguez-Paris ◽  
G Buczynski ◽  
J Cardelli
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Lines ◽  
Structure And Function ◽  
Dominant Negative ◽  
Contractile Vacuole ◽  
Vacuole Membrane ◽  
Endosomal Pathway ◽  
Transformed Cell Lines ◽  
And Function ◽  
Mutant Cells

The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in mutant cells, resulting in an accumulation in acidic smaller vesicles, probably lysosomes. Also, RabD N121I-expressing cell lines missorted a small but significant percentage of newly synthesized lysosomal alpha-mannosidase precursor polypeptides. However, the majority of the newly synthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway.

The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker protein

1999 ◽  
Vol 112 (22) ◽  
pp. 3995-4005 ◽  
Author(s):  
D. Gabriel ◽  
U. Hacker ◽  
J. Kohler ◽  
A. Muller-Taubenberger ◽  
J.M. Schwartz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Protein ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Contractile Vacuole ◽  
Specific Marker ◽  
Sharp Separation ◽  
Cell Fractions ◽  
Entire Network

The contractile vacuole system is an osmoregulatory organelle composed of cisternae and interconnecting ducts. Large cisternae act as bladders that periodically fuse with the plasma membrane, forming pores to expel water. To visualize the entire network in vivo and to identify constituents of the vacuolar complex in cell fractions, we introduced a specific marker into Dictyostelium cells, GFP-tagged dajumin. The C-terminal, GFP-tagged region of this transmembrane protein is responsible for sorting to the contractile vacuole complex. Dajumin-GFP negligibly associates with the plasma membrane, indicating its retention during discharge of the bladder. Fluorescent labeled cell-surface constituents are efficiently internalized by endocytosis, while no significant cycling through the contractile vacuole is observed. Endosomes loaded with yeast particles or a fluid-phase marker indicate sharp separation of the endocytic pathway from the contractile vacuole compartment. Even after dispersion of the contractile vacuole system during mitosis, dajumin-GFP distinguishes the vesicles from endosomes, and visualizes post-mitotic re-organization of the network around the nucleus. Highly discriminative sorting and membrane fusion mechanisms are proposed to account for the sharp separation of the contractile vacuole and endosomal compartments. Evidence for a similar compartment in other eukaryotic cells is discussed.

scholarly journals Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis.

1994 ◽  
Vol 127 (2) ◽  
pp. 387-399 ◽  
Author(s):  
R A Bacon ◽  
C J Cohen ◽  
D A Lewin ◽  
I Mellman
Keyword(s):  
Dictyostelium Discoideum ◽  
Membrane Trafficking ◽  
Development Program ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Cell Surface Receptor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Fluorescent Marker

We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indy1) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indy1 cells at the permissive temperature was poorly internalized in indy1 under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membrane-trafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indy1 cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indy1 mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning.

Trafficking of interleukin 2 and transferrin in endosomal fractions of T lymphocytes

1994 ◽  
Vol 107 (5) ◽  
pp. 1289-1295 ◽  
Author(s):  
V. Duprez ◽  
M. Smoljanovic ◽  
M. Lieb ◽  
A. Dautry-Varsat
Keyword(s):  
Horseradish Peroxidase ◽  
Interleukin 2 ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Recycling Endosomes ◽  
High Affinity ◽  
Human T Cell ◽  
Late Endosomes ◽  
Discontinuous Sucrose Gradient ◽  
Acidic Organelles

The T lymphocyte growth factor interleukin 2 binds to surface high-affinity receptors and is rapidly internalized and degraded in acidic organelles. The alpha and beta chains of high-affinity interleukin 2 receptors are internalized together with interleukin 2. To identify the intracellular pathway followed by interleukin 2, we have compared the subcellular distribution of interleukin 2, transferrin and a fluid-phase marker, horseradish peroxidase, in the human T cell line IARC 301.5. Transferrin was used as a marker of early and recycling endosomes, and horseradish peroxidase to probe for the whole endocytic pathway. Fractionation of intracellular organelles on a discontinuous sucrose gradient showed that internalized interleukin 2 is initially mostly found in compartments with similar densities to transferrin, e.g. early and recycling endosomes. The kinetics of entry and exit of interleukin 2 from such organelles was much slower than that of transferrin. Later on, interleukin 2 is predominantly found in dense lysosome-containing fractions. Very little, if any, interleukin 2 was found in fractions corresponding to late endosomes containing horseradish peroxidase. These results suggest that, after endocytosis, interleukin 2 enters early or recycling endosomes before it reaches dense lysosomes.

Rab22a affects the morphology and function of the endocytic pathway

2001 ◽  
Vol 114 (22) ◽  
pp. 4041-4049 ◽  
Author(s):  
Rosana Mesa ◽  
Cristina Salomón ◽  
Marcelo Roggero ◽  
Philip D. Stahl ◽  
Luis S. Mayorga
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Rab Proteins ◽  
Rab Protein ◽  
Late Endosomes ◽  
And Function ◽  
Negative Mutant

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

scholarly journals Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

1992 ◽  
Vol 103 (4) ◽  
pp. 1139-1152
Author(s):  
J.W. Kok ◽  
K. Hoekstra ◽  
S. Eskelinen ◽  
D. Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway ◽  
Golgi Network ◽  
Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi and contractile vacuole system and regulate osmoregulation

2016 ◽  
Vol 41 (2) ◽  
pp. 205-217 ◽  
Author(s):  
Katherine Maringer ◽  
Azure Yarbrough ◽  
Sunder Sims-Lucas ◽  
Entsar Saheb ◽  
Sanaa Jawed ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Contractile Vacuole

scholarly journals Enhancement of Ebola virus infection by seminal amyloid fibrils

2018 ◽  
Vol 115 (28) ◽  
pp. 7410-7415 ◽  
Author(s):  
Stephen M. Bart ◽  
Courtney Cohen ◽  
John M. Dye ◽  
James Shorter ◽  
Paul Bates
Keyword(s):  
Cell Lines ◽  
Ebola Virus ◽  
Amyloid Fibrils ◽  
Sexual Transmission ◽  
Fluid Phase ◽  
Infectious Individual ◽  
Transmission Potential ◽  
Western Africa ◽  
Ebov Infection

The 2014 western Africa Ebola virus (EBOV) epidemic was unprecedented in magnitude, infecting over 28,000 and causing over 11,000 deaths. During this outbreak, multiple instances of EBOV sexual transmission were reported, including cases where the infectious individual had recovered from EBOV disease months before transmission. Potential human host factors in EBOV sexual transmission remain unstudied. Several basic seminal amyloids, most notably semen-derived enhancer of viral infection (SEVI), enhance in vitro infection by HIV and several other viruses. To test the ability of these peptides to enhance EBOV infection, viruses bearing the EBOV glycoprotein (EboGP) were preincubated with physiological concentrations of SEVI before infection of physiologically relevant cell lines and primary cells. Preincubation with SEVI significantly increased EboGP-mediated infectivity and replication in epithelium- and monocyte-derived cell lines. This enhancement was dependent upon amyloidogenesis and positive charge, and infection results were observed with both viruses carrying EboGP and authentic EBOV as well as with semen. SEVI enhanced binding of virus to cells and markedly increased its subsequent internalization. SEVI also stimulated uptake of a fluid phase marker by macropinocytosis, a critical mechanism by which cells internalize EBOV. We report a previously unrecognized ability of SEVI and semen to significantly alter viral physical properties critical for transmissibility by increasing the stability of EboGP-bearing recombinant viruses during incubation at elevated temperature and providing resistance to desiccation. Given the potential for EBOV sexual transmission to spark new transmission chains, these findings represent an important interrogation of factors potentially important for this EBOV transmission route.

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