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Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi
and contractile vacuole system and regulate osmoregulation
KATHERINE MARINGER1,2,3 , AZURE YARBROUGH3,* , SUNDER SIMS-LUCAS2 , ENTSAR SAHEB3,4 ,
SANAA JAWED3,4 and JOHN BUSH3
1
Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine,
Pittsburgh, PA 15201, USA
2
Rangos Research Center, Children’s Hospital of Pittsburgh of UPMC,
Pittsburgh, PA 15224, USA
3
Biology Department, University of Arkansas at Little Rock, Little Rock, AR 72204-1099, USA
4
Biology Department, Baghdad University, Baghdad, Iraq
*Corresponding author (Email, [email protected])
Small-molecular-weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and is required for
protein transport from the ER to the Golgi complex; however, Rab2 has yet to be characterized in Dictyostelium
discoideum. DdRabS is a Dictyostelium Rab that is 80% homologous to DdRab1 which is required for protein
transport between the ER and Golgi. Expression of GFP-tagged DdRab2 and DdRabS proteins showed localization to
Golgi membranes and to the contractile vacuole system (CV) in Dictyostelium. Microscopic imaging indicates that the
DdRab2 and DdRabS proteins localize at, and are essential for, the proper structure of Golgi membranes and the CV
system. Dominant negative (DN) forms show fractionation of Golgi membranes, supporting their role in the structure
and function of it. DdRab2 and DdRabS proteins, and their dominant negative and constitutively active (CA) forms,
affect osmoregulation of the cells, possibly by the influx and discharge of fluids, which suggests a role in the function
of the CV system. This is the first evidence of GTPases being localized to both Golgi membranes and the CV system
in Dictyostelium.
[Maringer K, Yarbrough A, Sims-Lucas S, Saheb E, Jawed S and Bush J 2016 Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi
and contractile vacuole system and regulate osmoregulation. J. Biosci. 41 205–217] DOI 10.1007/s12038-016-9610-4
1.
Introduction
Rab proteins are the largest branch of the Ras superfamily of
small GTPases, and like other members, contain related
regions including at least four protein domains found in all
GTPases that are involved in the binding of GTP or GDP
(Nuoffer and Balch 1994; Harris et al. 2001). GTPases are
considered active in the GTP-bound state and inactive in the
GDP bound state (Nuoffer and Balch, 1994; Harris et al.
2001). All GTPases undergo a cycle mediated by guanine
nucleotide exchange factors (GEFs) and GTPase activating
Keywords.
proteins (GAPs) that stimulate exchange of GDP for GTP or
stimulate the hydrolysis of GTP (Nuoffer and Balch 1994;
Harris et al. 2001).
Vesicular transport is a complex biological process
involving the movement of membrane vesicles to and
from intracellular compartments. The secretory vesicular
pathway originates at the endoplasmic reticulum and
passes through the Golgi into lysosomes, secretory granules, or the extracellular environment via exocytosis
(Harris et al. 2001). The production, transport, and fusion of donor membrane vesicles with an acceptor
Contractile vacuole; DdRab2; DdRabS; Dictyostelium discoideum; Golgi
http://www.ias.ac.in/jbiosci
Published online: 13 May 2016
J. Biosci. 41(2), June 2016, 205–217, * Indian Academy of Sciences
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Katherine Maringer et al.
membrane compartment are regulated by numerous protein factors that work in an organized and specific fashion (Mellman 1996; Harris et al. 2001); Rab proteins are
one of the key groups of these proteins. The Rab family
includes more than 30 members that regulate vesicular
traffic between specific compartments of the endocytic
and exocytic pathways of eukaryotic cells (Nuoffer
1994; Simons 1993; Tisdale and Balch 1996). For example, DdRab4 (also named DdRabD) plays a role in
endocytosis and in the regulation of the structure and
function of the contractile vacuole complex (CV), possibly through membrane trafficking (Bush et al. 1996).
Dictyostelium discoideum is a simple eukaryotic amoeba often used to study the molecular mechanisms of
membrane trafficking in the endocytic pathway (Maniak
1999, 2001; Neuhaus and Soldati 2000; Rupper and
Cardelli 2001) due to its similarity to higher eukaryotic
cells (Harris et al. 2001). Like other protozoans,
D. discoideum utilizes a contractile vacuole complex
for the purposes of osmoregulation; specifically, it controls cell volume under hypotonic conditions. For the
first 20 min of exposure to a hypotonic environment,
the cell’s volume increases by approximately 20%, and
then it decreases regularly over the next 40 min
(Fountain et al. 2007). The CV cycle is regulated by
Rab proteins, their regulators, and their effectors. It is
enriched in membrane-associated alkaline phosphatase,
calmodulin (a Ca2+-binding protein), and vacuolar proton
pumps V-H-(+) ATPase (Nolta and Steck 1994). This
complex has a bipartite morphology that consists of a
bladderlike pump vacuole and an associated tubular
spongiomal network (Nolta and Steck 1994; Harris
et al. 2001). When the amoebae are confronted with a
hypotonic environment, water first enters the reticular,
spongiomal tubules of the CV system, which are rich in
RabD, and then activates DdRab11a. As the vacuoles
mature, Drainin is recruited to them; Drainin is a putative volume-sensing protein. The vacuoles are prepared
and tethered to the plasma membrane by DdRab8a and
disgorgin, a Rab GAP. The CV becomes polarized during a ring to patch transition, meaning that different
proteins become concentrated at the back or front of
the CV. A pore forms, and water is excreted
(Parkinson et al. 2014). Rab14 has also been localized
to the CV (Bush et al. 1994; Harris et al. 2001).
In concurrent studies, both DdRabS and DdRab2
showed an effect on growth and development. When
compared to the wild type, DdRab2-GFP-overexpressing cells (DdRab2-GFP) showed identical timing
and morphology of cells during development.
DdRab2(CA)-GFP (constitutive active) mutants formed
slightly larger aggregates and contained a larger number
of cells in each aggregate mound. After 24 h, the mound
J. Biosci. 41(2), June 2016
structures were smaller and very elongated, which
resulted in a greater number of small fruiting body
structures. DdRab2(DN)-GFP (dominant negative)
mutants had formed mound structures by 8 h, and after
16 h, they had formed fruiting body structures. After 24
h, the fruiting bodies were complete, and the soruus
appeared larger than that of the AX4 cells. DdRabSGFP overexpressing cells (DdRabS-GFP) failed to form
aggregates after 6 h, had begun to form very loose, small
aggregates by 8 h, and were unable to form fruiting
bodies and complete development. DdRabS(DN)-GFP
cells showed an increase in the rate of aggregation and,
after 8 h and had begun to form mound structures. By
24 h, DdRabS(DN)-GFP had completed development and
formed fruiting bodies (manuscript in progress).
All DdRab2-GFP cell lines and the DdRabS cell line
showed an increased growth rate when compared to the
AX4 cells. Interestingly, the DdRab2(DN)-GFP cell line
had the fastest growth rate at almost 10X that of the AX4
cells, while the RabS-GFP cells grew about twice as fast;
DdRabS(DN)-GFP mutants grew at a rate comparable to that
of AX4 cells (manuscript in progress).
In this study, we focussed on the intracellular localization
of the DdRab2 and DdRabS protiens. In other eukaryotic
systems, Rab2 has been found to localize to pre-Golgi intermediates and ER-Golgi membranes (Tisdale and Balch
1996). DdRabS is 80% similar to DdRab1 which has been
found to localize in ER-Golgi membranes (Bush et al. 1994).
We demonstrated that DdRab2 and DdRabS proteins localize to Golgi membranes as well as the CV system and effect
osmoregulation. This is the first evidence of Rab proteins
localizing to both Golgi membranes and the CV as well as
functioning in osmoregulation in Dictyostelium.
2.
2.1
Materials and methods
Cells and culture conditions
For all experiments, D. discoideum, wild-type strain AX4
(Dicty Stock Center, Chicago, IL, USA), pDneo2a-GFP,
D d R a b S - G F P , Dd R a b S ( D N ) - G F P , D d R a b 2 - G F P ,
DdRab2(DN)-GFP, and DdRab2(CA)-GFP expressing cell
lines were grown axenically at 21°C in shaking culture at
150 rpm in HL5 medium:1% oxoid proteose peptone, 1%
glucose, 0.5% yeast extract (Fisher Biotech, Fair lawn, New
Jersey, USA), 2.4 mM Na2HPO4, and 8.8 mM KH2PO4, pH
6.5. This media was supplemented with 300 mg/mL of
streptomycin sulfate and 100 mg/mL of ampicillin (Sigma,
St. Louis, MO, USA). Additionally, for the transfected cells,
HL5 medium was supplemented with 10 mg/mL of G418
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Rab proteins involved with Golgi and osmoregulation
(Invitrogen, Grand Island, NY, USA). To minimize background fluorescence from the HL5 medium, cells were incubated in ‘Loflo’ medium (1,000x FM salts 1, 1,000x FM
salts 2, 10,000x FM trace elements, glucose, 1 M K2HPO4,
casein peptone) for 24 h before the experiments (www.
dictybase.org).
2.2
Creation of GFP-tagged DdRabS and
DdRab2 cell lines
Dictyostelium RabS and Rab2 cDNA were subjected to
PCR using primers that had a SalI restriction enzyme on
the sense primer and an XhoI restriction enzyme on the
anti-sense primer. The resulting PCR products were ligated into the TA vector (Invitrogen, Grand Island, NY,
USA) and sequenced for both errors and the presence of
the SalI and XhoI sites. The DdRabS and DdRab2 PCR
TA products were then digested with SalI and XhoI,
purified, and ligated into the pDneo2a-GFP vector which
was also cut with SalI and XhoI. Resulting constructs
were sequenced for errors and reading frame confirmation. Constructs were then electrotransfected into
Dictyostelium AX4 cells and selected for with the G418
antibiotic (Invitrogen, Grand Island, NY, USA).
Dominant negative forms of the DdRabS and DdRab2
proteins, and the constitutively active form of the
DdRab2 protein, were created by changing the amino
acid asparagine (N) to isoleucine (I) at position 137 and
118 respectively (DN) and by changing the amino acid
glutamine (Q) to leucine (L) at position 64 (CA) using
t h e St r a t a g e n e Q u i c k C h a n g e ® I I S i t e - D i r e c t e d
Mutagenesis Kit (Stratagene, Santa Clara, CA, USA).
The resulting mutant constructs were sequenced to confirm the single changes in the proteins.
2.3
Western blotting
For Western blotting, 5×106 cells were harvested and resuspended in 1 mL of double-distilled water; the cells,
150 μL of fresh lysis buffer, and 0.3 gm of glass beads
were added to a 1.5 mL tube, and centrifuged for
10 min at 2200 rpm. 50 μL of the supernatant was
placed to a fresh 1.5 mL tube along with 50 μL of
fresh 2X SDS-loading dye. The sample was heat treated
for 10 min at 99oC, subjected to 10% SDS-PAGE, and
transferred to PVDF membrane (Millipore Cor. Bedford,
Cat. no IPVH00010) using a Hoefer Transfer Unit as
described (Bush et al. 1994). Blots were incubated with
primary antibodies (1: 2000 dilution of a mouse monoclonal anti-GFP antibody (Sigma, St. Louis, MO, USA)
in antibody buffer (20 mM Tris, pH 7.5, 140 mM NaCl,
0.05% Tween 20, 1% powdered milk). Samples were
then washed, incubated with goat anti-mouse secondary
antibody conjugated to horse radish peroxidase
(Phototope ® -HRP Western Blot Detection Kit, New
England Biolabs), and visualized by exposing the membrane to X-ray film for 60 s; the film was developed
according to manufacturers recommendations.
2.4
GFP visualization
Cells were cultured to a density of approximately 1–4×106
cells/mL, harvested, and allowed to settle on a glass cover
slip. Cell fluorescence was viewed and photographed using a
Nikon Eclipse 90i microscope equipped with 12V-100W
halogen lamp, external transformer, fly-eye lens built-in
and NCB11, ND8, ND32 filters. The Bright fields filter
and BrightLine® GFP Filter Set was used with 1000×
magnification.
2.5
Golgi visualization: Wheat germ Agglutinin staining
Wheat Germ Agglutinin (WGA) (Sigma, St. Louis, MO,
USA Aldrich) is a Golgi-specific stain (Clarke 2002). Cells
were harvested and allowed to settle on a glass cover slip. 50
μL of Texas Red-labeled WGA (1.0 μg/mL) was added to
the cells. The cells were incubated at 37oC for 10 min and
washed twice with Loflo medium. Fluorescence was visualized using the BrightLine® TXRED filter set, where the
Golgi appeared red, and the BrightLine® GFP filter was set
for comparison with GFP localization. Quantification was
performed using Fiji by ImageJ. The Pearson’s Correlation
Coefficient (PCC) was reported.
2.6
Contractile vacuole visualization: GFP visualization
and Calmodulin immunofluorescence
Cells were harvested and fixed on a glass cover slip.
Fluorescence was viewed and photographed at 1000× magnification using the BrightLine® GFP filter set on a Nikon
2000SE microscope and IPLab 3.7 software. Cells were
harvested and incubated at room temperature for 5 min in a
solution composed of 2% formaldehyde in PBS (pH 7.2),
0.1% DMSO and 1/3% protease peptone. Next, the cells
were chilled in methanol containing 1% formaldehyde at
−20°C for 5 min. Cells washed three times in PBS for
antibody staining (Bush et al. 1996; Fok et al. 1993;
Heuser et al. 1993) and then blocked with a 1:20 dilution
of normal goat serum (NGS) in a 0.1% BSA/PBS solution at
37°C for 30 min. The primary antibody, mouse antiCalmodulin, was diluted 1:400 in 0.1% BSA/PBS (Hulen
et al. 1991; Zhu et al. 1993). The cells were incubated with
anti-Calmodulin at 37°C for 30 min and washed three times
with PBS/0.5% Tween-20. The secondary antibody, RITC
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Katherine Maringer et al.
goat anti-mouse IgG (1 mg/mL), was diluted 1:200 in PBS/
0.1% BSA (Zhu et al. 1993) and incubated at 37°C for 30
min. Next, the cells were washed three times with PBS/0.5%
Tween-20, once with water, and then allowed to air-dry
(http://dictybase.org/techniques/). Calmodulin fluoresced
red when visualized using the BrightLine® TXRED filters
set. Comparisons were made to GFP localization, visible
under the BrightLine® GFP filter set.
To visualize the CV, cells were dyed with FM®1-43
(lipophilic styryl dye N-(3trietylammoniumpropyl)-4-(4(dibutylamino) styryl) pyridiniumdibromide (Molecular
Probes). First, cells were harvested and allowed to settle on
a glass cover slip. Next, FM®1-43 added for a final concentration of 16 μM in KK2 phosphate buffer (1.6 mM KH2PO4
and 40 mM K2HPO4) and chilled for 1 min on ice. Cells
were washed then visualized using the BrightLine® GFP
filters set and the BrightLine ® TXRED filters set.
Quantification was performed as previously described.
2.7
Inhibitors
Brefeldin A (BFA) is a fungal metabolite that disrupts the
structure and function of the Golgi apparatus (Sigma, St.
Louis, MO, USA 2003). First, cells were harvested and
allowed to settle in a 35×10 mm Petri dish. Loflo medium
containing BFA at a concentration of 5 μM was added. Cells
were incubated for 60 min. Fluorescence from GFP and
FM®1-43 (AX4) was viewed and photographed using the
BrightLine® GFP filters set.
Concanamycin A (CMA) is an ATPase inhibitor specific
to the CV (Temesvari et al. 1996a, b). Inhibition effects on
the different cell lines were studied at isotonic, hypotonic,
and hypertonic environments. First, cells were harvested and
allowed to adhere to a glass cover slip. Loflo medium, water,
or 100 mM sucrose in Loflo medium was added, respectively. CMA (Sigma, St. Louis, MO, USA) was added to all
treatments at a final concentration of 5 μM. Additionally,
AX4 cells were treated for 1 min on ice with the dye FM®143 for visualizing the contractile vacuole. Fluorescence from
either GFP or FM®1-43 was viewed and photographed using
the BrightLine® GFP filter set.
2.8
Osmotic stress assay
The contractile vacuolar network was visualized under the
microscope using the BrightLine® filter set. Comparisons
were made for cells in an isotonic, hypotonic, and hypertonic
environment. Cells were incubated for 60 min in HL5, water,
or 100 mM sucrose in HL5 medium respectively. The cells
were harvested and allowed to settle on a glass cover slip,
viewed, and photographed.
J. Biosci. 41(2), June 2016
3.
3.1
Results
Construction of DdRabS- and DdRab2-overexpressing and mutant cell lines
The DdRabS-GFP cell line was constructed, harvested, and
subjected to SDS-PAGE and Western blot analysis as mentioned above. Expression of DdRabS-GFP was confirmed by
Western blot at approximately 53 kDa (DdRabS 25 kDa +
GFP 28 kDa). The DdRab2-GFP cell lines were constructed
in the same manner and confirmed using Western blot
(DdRab2 23 kDa + GFP 28 kDa = 51 kDa).
DdRabS-GFP and DdRab2-GFP were mutated to change
an encoded asparagine to an isoleucine as described above.
This amino acid plays a critical role in the binding of GTP
and GDP, and comparable mutations in other Rab proteins
have resulted in the formation of proteins that function in a
DN manner (Walworth et al. 1989; Tisdale et al. 1992; Bush
et al. 1996). The mutated RabS-GFP and DdRab2-GFP
plasmids were transformed into Dictyostelium AX4 cells
and selected using G418. Western blots confirmed expression of the DN DdRabS-GFP, and DdRab2-GFP proteins at
approximately 53 and 51 kDa.
DdRab2-GFP was mutated by replacing the glutamine
with leucine at position 64 to generate a constiutively active
protein, which preferntially binds to GTP, as described. The
mutated DdRab2-GFP plasmid was also transformed, selected for using G418, and expression confirmed with Western
blot of the constitutively active DdRab2-GFP protein at
approximately 51 kDa.
3.2 Dictyostelium RabS and Rab2 colocalize with both
Golgi membranes and the contractile vacuole system
Immunofluorescence microscopy was used to determine the
intracellular location of both the DdRab2 and DdRabS proteins. Cell lines over-expressing GFP-tagged DdRab2-GFP,
DdRab2(CA)-GFP, DdRab2(DN)-GFP, DdRabS-GFP, and
DdRabS(DN)-GFP were subjected to fluorescence microscopy to infer the possible location. As a control, we
expressed free GFP in AX4 and found it evenly distributed
throughout the cell (not shown). All cell lines showed areas
of GFP fluorescence in areas thought to be Golgi membranes
and the CV system (figure 1).
To confirm the localization as Golgi membranes (figure 1), all cell lines were subjected to fluorescence microscopy following staining with the known Dictyostelium Golgi
marker WGA (Zhu et al. 1993) (figure 2). RabS-GFP- and
DdRabS(DN)-GFP-expressing cells show colocalization
with WGA. However, the Golgi appears to be fractionated
and dispersed throughout the cell in the DdRabS(DN)-GFP
cell line when compared to the AX4 cells. This apparent
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Rab proteins involved with Golgi and osmoregulation
Figure 1. Localization analysis of GFP-labelled DdRabS and Rab2 suggest these proteins are associated with the Golgi and contractile
vacuole membranes. (A-F) GFP images of WT-AX4, DdRabS-GFP, DdRabS(DN)-GFP, DdRab2-GFP, DdRab2(CA)-GFP, and
DdRab2(DN)-GFP cell lines. (A) WT-AX4 control cells with no GFP expression. (B) RabS-GFP cells show a strong GFP fluorescence
in a spot which may be the Golgi (indicated by arrows) and GFP fluorescence around some vesicles which may be contractile vacuoles
(indicated by arrows). (C) DdRabS(DN)-GFP-expressing cells showing areas of GFP fluorescence in small ‘spots’ throughout the cell
(indicated by arrows) which may be subparts or fractionation of the Golgi. (D) DdRab2-GFP cells with strong GFP fluorescence in small
‘spots’ throughout the cell (indicated by arrows) which may be Golgi elements as well as GFP fluorescence surrounding the visible CV
network (indicated by arrows) which may be the spongiomal network of the CV system. (E) DdRab2(CA)-GFP-expressing cells showing
several areas of intense GFP expression which may be Golgi (indicated by arrows); however, it does not appear to be fractionated as in the
DdRab2-GFP cells. The spongiomal network of the CV system is still apparent in the DdRab2(CA)-GFP cell line. (F) Image DdRab2(DN)GFP cells showing some spots of GFP expression which may be Golgi vesicles (indicated by arrows) as well as some contractile vacuoles
which are interestingly centrally located within the cell.
fractionation is also seen in the DdRab2(DN)-GFP cell line.
DdRab2-GFP, DdRab2(CA)-GFP, and DdRab2(DN)-GFP
cell lines all show partial colocalization with the WGAstained Golgi membranes. The quantification of this colocalization supported these findings with the Pearson correlation coefficient values of 0.577 for DdRabS(DN)-GFP,
0.379 for DdRab2(CA)-GFP, 0.293 for DdRab2-GFP,
0.265 for DdRabS-GFP, and 0.192 for DdRab2(DN)-GFP
(figure 2). This colocalization was quantified as previously
described.
It has been demonstrated that calmodulin is localized
to the contractile vacuole (CV) in Dictyostelium (Heuser
et al. 1993; Zhu et al. 1993; Bush et al. 1994; Harris
et al. 2001). Immunofluorescence microscopy was used
to determine if the GFP-tagged DdRabS or DdRab2
proteins colocalized with the established CV marker
antigens (figure 3). DdRabS-GFP and DdRabS
(DN)-GFP cells appear to partially colocalize with the
CVs. The DdRab2-GFP, DdRab2(CA)-GFP, and
DdRab2(DN)-GFP cells all appear to colocalize with
the spongiomal network of the CV system rather than
the vacuoles themselves. The quantification of this
colocalization supported these finding with correlation
coefficient values of 0.852 for DdRab2(CA)-GFP,
0.815 for DdRab2-GFP, 0.749 for DdRab2(DN)-GFP,
0.666 for DdRabS(DN)-GFP, and 0.5589 for DdRabSGFP (figure 3). This colocalization was quantified as
mentioned above. This data confirms localization of the
DdRabS and DdRab2 proteins to both Golgi membranes
and the CV system.
3.3
DdRab2 proteins redistribute to the ER in
the presence of BFA
Brefeldin A (BFA) is a fungal metabolite that induces
Golgi proteins to redistribute into the ER and blocks
their secretion from the Golgi apparatus (Donaldson
et al. 1991). Rab2 has been previously reported to be
a resident of pre-Golgi intermediates and to participate
in the segregation of anterograde and retrograde transported proteins following export from the ER; it is also
required for protein traffic between the ER and the
Golgi complex (Tisdale and Balch 1996). To determine
J. Biosci. 41(2), June 2016
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Katherine Maringer et al.
Figure 2. Colocalization of GFP-labelled Rabs with Texas Red-labelled Wheat Germ Agglutinin suggest a partial association with Golgi
membrane and Rab over-expressing and mutant cell lines. (A-F) Images of RabS-GFP and DdRab2-GFP over-expressing and mutant cell
lines stained with known Golgi marker Wheat Germ Agglutinin (WGA) (shown in red) with GFP fluorescence (shown in green), and
merged images (shown in yellow). Cells were allowed to adhere to glass coverslips and were incubated at 37oC for 10 min with 1μg/mL
WGA. (A) WT-AX4 cells showing the WGA stained Golgi (indicated by arrows). (B) RabS-GFP cells showing the WGA stained Golgi in
red (indicated by arrows), and the DdRabS-GFP in green (indicated by arrows). The merged image show colocalization of the GFP with the
WGA stained Golgi (yellow) indicating Golgi localization. (C) DdRabS(DN)-GFP cells show overlap with Golgi stained membranes
(indicated by arrows). The Golgi appears fractionated in the DdRabS(DN)-GFP cell line. (D) DdRab2-GFP cells show colocalization with
the WGA stained Golgi (indicated by arrows); however, this localization appears to be partial as there are some areas that do not overlap.
(E-F) DdRab2(CA)-GFP and DdRab2(DN)-GFP cells show partial overlap with the WGA stained Golgi. There are, however, some areas of
GFP fluorescence not colocalized with Golgi membranes indicating only partial localization. In the DdRab2(CA)-GFP and DdRab2(DN)GFP cells, the Golgi also appears to be diffused throughout the cell instead of in one spot as in the control and DdRab2-GFP cell lines.
if the DdRabS and DdRab2 proteins localize to ERGolgi membranes, cells were treated with Brefeldin A
as previously described and viewed using fluorescence
microscopy. Figure 4 shows a strong redistribution of
the GFP signal from the Golgi to what may be the ER
in the DdRab2-GFP, CA, and DN cell lines. The CA
and DN forms of the DdRab2 protein show possible
fractionation of the ER under BFA inhibition. These
results indicate that DdRab2 proteins may localize between ER and Golgi membranes.
J. Biosci. 41(2), June 2016
3.4 DdRabS- and DdRab2-over-expressing and mutant
cell lines are defective in osmoregulation when exposed
to osmotic stress
DdRabS and DdRab2 were found to be partially localized to
the CV system. This localization suggests that they may
potentially play a role in its function. To test this possibility,
cells were collected by centrifugation and re-suspended in
hypo-osmotic (100% water) or hyperosmotic (100 mM sucrose in HL5) medium for 1 h, allowed to settle on glass
Rab proteins involved with Golgi and osmoregulation
211
Figure 3. Colocalization of GFP-labelled Rabs with Texas Red-labelled Calmodulin (CV marker) indicates a partial association with the
CV systems and Rab over-expressing and mutant cell lines. (A-E) Merged images of GFP fluorescence (green) and calmodulin antibody
immunofluorescence (red) show overlapping areas (yellow-orange) where colocalization occurs. (A, B) DdRabS-GFP over-expressing and
DdRabS(DN)-GFP cells show partial colocalization with the CV network. (C-E) DdRab2-GFP, DdRab2(CA)-GFP, and DdRab2(DN)-GFP
cells show partial colocalization with the CV system. In these DdRab2 cell lines, the colocalization appears to be with the spongiomal
network of the CV system rather than the vacuoles.
coverslips for 10 min, and viewed under brightfield microscopy
(figure 5). As a control, cells were also photographed after
incubation in HL5 medium. Cells exposed to isotonic (HL5)
conditions were able to intake and expel the medium normally. It
should be noted, however, that the DdRab2-GFP and
DdRab2(CA)-GFP (figure 5, panel 1B and 1C) cells appeared
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Figure 4. Brefeldin A (BFA) treatment resulted in redistribution of the GFP-tagged DdRabS and DdRab2 over-expressing and mutant cell
lines. (A-F) Fluorescence microscopy of cells incubated for 60 min in the presence of 5 μM Brefeldin A Golgi inhibitor. (A) WT-AX4 cells
stained with FM®1-43 dye and treated with BFA. (B) DdRabS-GFP over-expressing cells show strong GFP fluorescence indicating redistribution of the Golgi localized Rab into the ER (indicated by arrows). (C) DdRabS(DN)-GFP cells show strong GFP fluorescence in
several spots throughout the cell indicating disruption of the Golgi and redistribution to the ER. (D-F) DdRab2-GFP, DdRab2(CA)-GFP,
and DdRab2(DN)-GFP cells show partial redistribution of the Golgi-associated Rabs to the ER and disruption of the Golgi.
to have a greater number of CVs present (approximately double)
compared to the AX4 cells; this did not appear to have an effect
on osmoregulation under these conditions.
Cells exposed to hypo-osmotic conditions became rounded
and swollen (figure 5, panel 2). In the DdRab2-GFP and
DdRab2(CA)-GFP cells, the CVs appeared to have fused into
one large CV, indicating that these cells were not able to expel
the water causing accumulation and fusion of the CVs. These
cells also lysed due to water intake after 60 min. The
DdRab2(DN)-GFP cells were swollen and there were no CVs
present. The lysing seen in the DdRab2-GFP and DdRab2(CA)GFP cells was not seen in DdRab2(DN)-GFP cells. Although
CVs were not seen in this cell line, they were able to regulate
water intake similarly to the AX4 cells. Converse to the
DdRab2-GFP and DdRab2(CA)-GFP cells, DdRabS-GFP cells
appeared similar to AX4 cells; however, they did not lyse under
these hypo-osmotic conditions. DdRabS-GFP cells were able to
live and regulate the hypo-osmotic environment longer than the
AX4 cells. Similar to the DdRab2(DN)-GFP cells,
DdRabS(DN)-GFP cells did not appear to have any CVs present, and they functioned similarly to AX4 cells.
Cells exposed to hyperosmotic conditions had shrunk in
size, and sucrose-containing vacuoles (sucrosomes) were
evident. DdRab2-GFP and DdRab2(CA)-GFP cells
appeared to have a greater number of sucrosomes when
compared to AX4 cells. All DdRab2-GFP cell lines
contained a bulging cytoplasm, and it appeared that all
J. Biosci. 41(2), June 2016
cellular organelles were concentrated centrally within the
cells. DdRabS-GFP and DdRabS(DN)-GFP cells were different in morphology compared to AX4 cells; they
appeared to have swollen slightly and became rounded in
shape, and the sucrosomes were larger than those of the
AX4 cells. These results suggest that DdRabS and
DdRab2 function in osmoregulation. The opposite effects
seen in the hypo-osmotic environment with DdRab2 and
DdRabS cells indicates that although they both function in
osmoregularity, they play distinctly different roles.
3.5 Concanamycin A (CMA) treatment induces differential
CV Structural changes in both DdRabS- and DdRab2-overexpressing as well as DdRabS and DdRab2 mutant cell lines
Concanamycin A (CMA) is a specific V-H+-ATPase inhibitor (Kinashi et al. 1984). In vitro, CMA is an extremely
potent inhibitor (IC50= 1 to 5 nm) of V-H+-ATPases, and it
is able to discriminate between mitochondrial, plasma membrane, and vacuolar ATPases (Bowman et al. 1988;
Mattsson et al. 1991; Woo et al. 1992; Temesvari et al.
1996a, b). CMA has been shown to inhibit CV function
and induce gross morphological changes in D. discoideum
cells (Temesvari et al. 1996a, b). DdRabS and DdRab2 show
partial localization to the CV network, and we have shown
them to be involved in osmoregulation. To examine the
Rab proteins involved with Golgi and osmoregulation
213
Figure 5. DdRabS and DdRab2 over-expressing and mutant cell lines show defects in osmoregulation under osmotically stressed
conditions (hypo- and hypertonic solutions.) (A-F) AX4 (A), DdRab2 (B), DdRab2(CA)-GFP (C), DdRab2(DN)-GFP (D), DdRabSGFP (E), and DdRabS(DN)-GFP (F) cellular appearance after 60 min incubation under osmotic stress conditions. Panel 1 A-F: Cells under
isotonic conditions were incubated in HL5 medium. DdRab2-GFP (1B), DdRab2(CA)-GFP (1C), and DdRabS-GFP (1E) cells appear to
have more contractile vacuoles present compared to the AX4 (A) cell line. Both DdRab2(DN)-GFP (1D) and DdRabS(DN)-GFP (1F) cells
appear to have diminished CVs. Panel 2 A-F: Cells under hypotonic conditions were incubated in water. DdRab2-GFP (2B) and
DdRab2(CA)-GFP (2C) cells appear to have a single very large contractile vacuole indicating that the contractile vacuoles have fused
into one and the cell is deficient in water release. These cells lysed sooner than the WT-AX4 cells confirming deficient water release.
DdRabS-GFP (2E) cells appear similar to AX4 cells; however, these cells did not lyse due to excess water after significant time as the AX4
cells do. DdRab2(DN)-GFP (2D) and DdRabS(DN)-GFP (2F) cells do not appear to have any contractile vacuoles present; however, the
cells are swollen from the hypotonic environment. The lack of CVs present indicates that the cells have expelled the excess water unlike the
CA and over-expressing cell lines. Panel 3 A-F: Cells under hypertonic conditions were incubated in HL5 + 100 mM sucrose. DdRab2-GFP
(3B) and DdRab2(CA)-GFP (3C) cells appear to have a larger number of sucrose containing vacuoles (sucrosomes) compared to the WTAX4 cells. The DdRab2(DN)-GFP (3D) cells do not appear to have any sucrosomes; however, they have some ballooning protrusions
extending from the cell. DdRabS-GFP (E) cells do not appear to contain any sucrosomes; however, they have ballooning protrusions
extending from the cells. DdRabS(DN)-GFP (F) cells appear similar to the WT-AX4 cells under hypertonic conditions.
effects of CMA, cells were incubated with 5 μM CMA in
either HL5, 100% water, or 100 mM sucrose + HL5 for 60
min; they were then allowed to adhere to glass coverslips
and veiwed using immunoflourescence microscopy. AX4
cells were stained with FM-143® vital dye to visualize the
vacuoles after CMA treatment. Figure 6 panel 1A–F shows
cells in an HL5, panel 2 A–F shows cells in 100% water, and
panel 3 A–F shows cells in 100 mM sucrose + HL5.
Dissasociation of the sprongiomal network is evident in the
AX4 cell line under all conditions.
Under isotonic conditions, DdRab2-GFP cells show
very few diminished vacuoles, and the spongoimal network appears completely diffused (figure 6, panel 1B).
In DdRab2(CA)-GFP cells, the spongiomal network
appears to still be intact, and there is one large vacuole
present in the cell indicating that all CVs have fused
into one (figure 6, panel 1C). The spongiomal network
also appeared intact in DdRab2(DN)-GFP cells; however, no vacuoles were present (figure 6, panel 1D).
Conversly, in RabS-GFP cells, the spongiomal network
is not evident but vacuoles are. Also, the RabS-GFP-
enriched Golgi appears to be fractionated (figure 6, panel 1E). This fractionated Golgi is also apparent in
DdRabS(DN)-GFP cells, and the CV system appears
completely diminished (figure 6, panel 1F).
Under hypotonic conditions, all cells have swollen and
become rounded. DdRab2-GFP and DdRab2(CA)-GFP cells
appear to have few diminished vacuoles, and the DdRab2enriched membranes appear to have fused into one (figure 6,
panel 2B-C). DdRab2(DN)-GFP cells also show fusion of
membranes into one vacuole; however, the spongiomal network is still evident in these cells (figure 6, panel 2D).
Conversly, RabS-GFP cells show a large number of vacuoles
present throughout the cell and fractionation of DdRabS
associated Golgi membranes (figure 6, panel 2E).
Fractionation is also evident in DdRabS(DN)-GFP cells (figure 6, panel 2F).
Under hypertonic conditions, all of the cells will shrink.
In DdRab2-GFP and DdRab2(CA)-GFP cells, some sucrosomes were present, and DdRab2 enriched membranes were
fused into one (figure 6, panel 3B-C). DdRab2(DN)-GFP
cells also have fewer sucrosomes; however, the fused GFP
J. Biosci. 41(2), June 2016
214
Katherine Maringer et al.
Figure 6. Concanamycin A (CMA), a V-H+-ATPase inhibitor, induces differential CV structural changes in DdRab2 and DdRabS overexpressing and mutant cell lines as compared to wild-type cells. Distribution of GFP in the DdRab2-GFP (B), DdRab2(CA)-GFP (C),
DdRab2(DN)-GFP (D), DdRabS-GFP (E), and DdRabS(DN)-GFP (F) cell lines and FM®1-43 stained AX4 (A) cells treated for 60 min
with 5 μM CMA. Panel 1 A-F: Cells treated in Loflo medium (isotonic). Disassociation of the spongiomal network of the CV system is
evident in all cell lines. In the DdRab2(CA)-GFP cell line, a large CV is shown centrally located within the cell. Panel 2 A-F: Cells treated
in water (hypotonic). The CV system appears completely diminished in all mutant cell lines. The large contractile vacuoles seen in the
DdRab2-GFP over-expressing and DdRab2(CA)-GFP cell lines in Figure 8 Panel 2 B and C are not present in cells treated with CMA.
These cells also did not lyse sooner than the controls. There is an intense are of GFP fluorescence throughout the Golgi in all mutant cell
lines. Panel 3 A-F: Cells treated in HL5 + 100 mM sucrose (hypertonic). Little disassociated CV network can be seen due to the water lost
by cells in the hypertonic environment. The Golgi vesicles can still be seen in all mutant cells; however this is diminished due to
dehydration of the cells.
enriched area seen in the over-expressor and CA cells is not
present in the DN form. Instead, a very diffused area of GFP
can be seen, possibly the spongiomal network or diffused
ER-Golgi membranes (figure 6, panel 3D). In RabS-GFP
cells, fractionation of the Golgi is no longer evident, and
no sucrosomes can be seen (figure 6, panel 3E). Similarily,
DdRabS(DN)-GFP cells no longer show fractionation of ERGolgi membranes (figure 6, panel 3F). These results support
the evidence for separate roles for DdRab2 and DdRabS in
osmoregulation.
4.
Discussion
We have demonstrated that DdRabS and DdRab2 proteins
are localized to ER-Golgi membranes and the CV, and that
these proteins function in the osmotic regulation of the cell.
Rab2 has previously been found to be a resident of pre-Golgi
intermediates and to participate in the segregation of anterograde and retrograde transported proteins following export
from the ER (Chavrier et al. 1990; Aridor et al. 1995). It has
also been previously demonstrated (Tisdale et al. 1992;
Tisdale and Balch 1996) that Rab2 is required for protein
traffic between the ER and Golgi complex (Tisdale and
Balch 1996). Our analysis using WGA staining demonstrated that DdRab2 and DdRabS were localized to ER-Golgi
J. Biosci. 41(2), June 2016
membranes as predicted. Interestingly, the distribution of the
GFP-tagged DdRabS and DdRab2 in the Golgi differed;
DdRabS-GFP appears to be more enriched in the Golgi than
DdRab2-GFP, and while DdRab2-GFP localized to the
Golgi, it was less concentrated DdRabS-GFP. In the DN
lines and DdRab2(CA), the Golgi appears fractionated but
colocalization is still apparent. When cells were treated with
BFA (Donaldson et al. 1991), there was a strong area of GFP
fluorescence where the DdRab2 and DdRabS proteins had
appeared to redistribute to the ER. In DN and CA forms of
DdRab2 and DdRabS, fractionation of the ER was evident,
suggesting that these proteins are crucial for normal function
and transport between these organelles.
Immunofluorescence using anti-Calmodulin, a CV marker, (Harris et al. 2001) revealed partial colocalization of
DdRab2 and DdRabS proteins to the spongiomal network
of the CV system. Similar to our colocalization studies of
DdRabS-GFP and DdRab2-GFP to the Golgi, the distribution of DdRabS-GFP and DdRab2-GFP within the CV network differed. DdRab2-GFP appeared to be more enriched
in the CV network than DdRabS-GFP, whereas DdRabSGFP appeared to be more enriched in the Golgi than
DdRab2-GFP. Dictyostelium rabs DdRab11 and DdRabD
are enriched in the CV system and have been shown to
regulate the structure and function of this organelle (Harris
215
Rab proteins involved with Golgi and osmoregulation
et al. 2001). Our data demonstrates that these proteins not
only colocalize to the CV but also have a role in its function.
The CVs of cells exposed to varying osmotic conditions
were unable to properly expell their contents in DdRab2GFP, DdRab2(CA)-GFP, and RabS-GFP cell lines; in the
DN forms of these proteins, the condition appeared reversed.
DdRab2(DN)-GFP and DdRabS(DN)-GFP appeared to have
very few if any CVs; however, the cells still appeared functional. This may indicate that they are over-expelling their
contents; since the CVs are constantly fusing with the membrane to expell, they are not visible.
This affect on osmoregulation could be due to DdRab2 and
DdRabS effecting the novel DdRab8A-GAP, Disgorgin, which,
along with Drainin and its regulator DdRab11A, control CV
discharge. Drainin and Disgorgin/DdRab8A sequentially localize to the CV membrane at the late charging stage and control
different stages in the process (Du et al. 2008). In DN forms of
DdRab2 and DdRabS, there may be an efflux of Disgorgin and
Drainin causing hyper-expulsion of the CVs, resulting in the
observed phenotype. A role in osmoregulation for the DdRab2
and DdRabS proteins is supported by the dissasociation of the
spogiomal network and loss of CV function in cells treated with
CMA. In Dictyostelium, CMA inhibits CV function and induces
gross morphological changes in cells, characterized by a decrease in small endo-lysosomal vesicles and the formation of
large intracellular vacuoles containing fluid phase and contractile
vacuole markers (Temesvari et al. 1996a, b). In the presence of
CMA, all cell lines appeared to have diminished CVs, and the
disociation of the spongiomal network was evident. CMA inhibits proton pump actions (Temesvari et al. 1996a, b); in a hypoosmotic environment, AX4 cells treated with CMA swelled
rapidly and ruptured. DdRab2, DdRab2(CA), and RabS-GFP
cells appeard to reverse this phenotype, further supporting their
role in CV funtion. Interestingly, both DN cell lines appeared to
have an intact spongiomal network and behaved similiarily to the
wild type in the presence of CMA. We suggest that DdRab2 and
DdRabS play a role in membrane trafficking steps of different
subcompartments in the CV system possibly by affecting the
trafficking of proton pumps.
In Dictyostelium, CV-resident proteins enter the secretory
pathway and are transported through the Golgi apparatus
before arriving at the CV (Mercanti et al. 2006). Our results
suggest dual localization of DdRab2 and DdRabS proteins to
ER-Golgi membranes and the CV system, indicating a potential role in their function by regulating vesicle transport
between and/or within them. It has been previously reported
that an α-Kinase is localized to the CV and Golgi in
Dictyostelium, and it functions in membrane trafficking/fusion. Furthermore, it was speculated that this α-Kinase may
act via GTPases to alter myosin filament assembly control in
a manner that blocks normal contractile ring formation and
contraction (Betapudi et al. 2005); however, this specific
dual localization/function has not been reported specifically
for Rab GTPases in Dictyostelium. This dual localiztion/
function is supported by the CV being a post-Golgi compartment indicated by O-glycosylated proteins in its membranes (Gabriel et al. 1999; Gerisch et al. 2002). It also
shares some membrane proteins with the endo-lysosomal
system another post Golgi compartment.
Although dual localization to ER-Golgi and CV systems of
Rab GTPases has not been reported, several other cases of dual
localization of Rabs have been demonstrated. Some examples
include hsRab9b (Lombardi et al. 1993; Yoshimura et al.
2007; Hutagalung and Novick 2011), Rab12 in Wistar rats
(Iida et al. 1996, Olkkonen et al. 1993, Hutagalung and
Novick 2011), hsRab13 (Zahraoui et al. 1994; Hutagalung
and Novick 2011), and hsRab14 (Junutula et al. 2004;
Larance et al. 2005; Hutagalung and Novick 2011). Our data
suggests a novel pathway for Dictyostelium Rab trafficking
between ER-Golgi membranes and the CV system. Vacuolar
proton pumps are present in the Golgi and both the CV system
and the endo-lysosomal system in Dictyostelium (Temesvari
et al. 1994; Clarke and Heuser 1997; Neuhaus et al. 1998;
Gerisch et al. 2002). It has been suggested that GTPases may
traffic the V-ATPase complex (Dieckmann et al. 2012). It is
possible that DdRab2 and DdRabS are trafficking vacuolar
proton pumps from ER-Golgi-CV, and possibly the endolysosomal system as well. The CV network and the endosomal
pathway have been linked (Temesvari et al. 1996a, b), so the
involvement of DdRab2 and DdRabS along this pathway will
need to be explored as well. Furthermore, DdRabS appears
more enriched in the Golgi than the CV, whereas DdRab2
appears more enriched in the CV than the Golgi. If these Rabs
are functioning in trafficking of proton pumps, their direction
in trafficking may contribute to their differing localization
concentrations. Further experiments will be needed to show
the exact role of DdRab2 and DdRabS in ER-Golgi-CV
transport.
Acknowledgements
We would like to acknowledge the University of Arkansas at
Little Rock for funding this study and providing the resources and equipment to undertake them. We would also like to
acknowledge University of Pittsburgh School of Medicine
for their assistance in getting this document ready for publication. We would also like to acknowledge Dictybase: it has
been a valuable tool, not only for strains and plasmids, but as
a source of information and protocols.
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MS received 01 September 2015; accepted 15 April 2016
Corresponding editor: STUART A NEWMAN
J. Biosci. 41(2), June 2016