FIGURE LEGENDS
FIGURE 1. Mitochondria morphology remains tubular after ESAT-6
expression in A549 AECs. ( A) A549 cells were transfected with
pcDNA3.1-ESAT-6-myc for 48 hours prior to fixation and processing with
anti-myc antibodies (ESAT-6, red) and anti-COXIV (mitochondria,
green)(top panel). Mitochondrial morphology in cells expressing ESAT-6
appears similar to untransfected cells. For comparison, A549 cells were
infected with Mtb and cells fixed and stained for mitochondria using
Mitotracker Red® (lower panel in A), as in (Fine-Coulson et al. ,
2015). After infection with Mtb, mitochondrial morphology appears
concentrated and fragmented. Scale bar = 10 µm. (B) The percentage of
cells with tubular, intermediate or fragmented mitochondria was
determined in cells transfected with ESAT-6-myc or with pcDNA3-mEmerald
as a transfection control. N=3, >100 cells counted per
transfection. PE17 does not localize to the mitochondria of
A549 cells but does decrease mitochondrial mass. ( C) AECs were
transfected with pcDNA3.1-PE17-myc-his6 48 hours prior to fixation and
processing. Cells were probed with anti-COXIV (mitochondria,
green), anti-myc (PE17, red), and stained with DAPI (nucleus, blue).
Images were captured by a confocal microscope and are representative of
three independent experiments. Arrows highlight inclusions apparent in
the transmitted light images that correspond to PE17 positive
structures. Scale bar = 20 µm. (D) PE17 (red) appears as clusters of
empty spheres adjacent to the nucleus (blue). (E) Images captured from
the slides prepared in (C) were analyzed for organelle mass using an
in-house FIJI macro. Over 130 cells between three slides were measured
for the percent area of the cell containing mitochondria for each
condition. N = 3. Error bars indicate SEM. Significance was measured
using a Mann-Whitney U test. ***Indicates p-values <
0.0001.
FIGURE 2. Expression of PE17 in A549 AECs alters Golgi stack and
trans-Golgi network, but not ER, morphology and mass. AECs were
transfected with pcDNA3.1-PE17-Myc-His6 48 hours prior to fixation and
processing. A,C,E) Cells were probed with
anti-myc (PE17), anti-calnexin (ER; A), anti-Gpp130 (golgi stack; C),
anti-TGN46 (trans Golgi network; E) and stained with DAPI
(nucleus). Images were captured on a confocal microscope and are
representative of three independent experiments. Scale bar = 20 µm. B,
D, F) Images captured from the slides prepared in A,C,E,
respectively, were analyzed using in-house FIJI macros. Over 90 cells
between three slides were measured for the percent area of the cell
containing the respective organelle for each condition. Error bars
indicate SEM. Significance was measured using a Mann-Whitney test.
***Indicate p-values < 0.0001. Values >0.05 were
considered not significant.
FIGURE 3. PE17 expression does not cause disruptions in the
cytoskeleton. AECs were transfected with
pcDNA3.1-PE17-myc-His6 48 hours prior to fixation and
processing. Cells were stained with DAPI (nucleus) and phalloidin
(F-actin; A) and probed with anti-myc (PE17) and anti-alpha
tubulin (microtubules; B). Images were captured by a confocal
microscope and are representative of two independent experiments. Scale
bar = 25 µm.
FIGURE 4. Expression of PE17 in A549 AECS does not alter the
mass of recycling or early endosomes but alters the mass of late
endosomes and lysosomes. AECs were transfected with pcDNA3.1-PE17 48
hours prior to fixation and processing. (A,C,E,G) Cells were probed with
anti-myc (PE17), anti-transferrin receptor (recycling endosomes; A),
anti-EEA1 (early endosomes; C), anti-CD63 (late endosomes; E),
anti-lamp1 (lysosomes; G) and stained with DAPI (nucleus). Images were
captured on a confocal microscope and are representative of three
independent experiments. Scale bar = 20 µm. (B, D, F, H) Images
captured from the slides prepared in A,C,E,G, respectively, were
analyzed using in-house macros in FIJI. Over 90 cells in B, D, and H and
over 60 cells in E were measured between experiments for each condition.
Error bars indication SEM. Significance was measured using a
Mann-Whitney U test. ***Indicate a p-values < 0.001. Values
>0.05 were considered nonsignificant.
FIGURE 5. PE17 localizes to lipid droplets via the C-terminal
domain. AECs were transfected with pcDNA3.1-PE17-Myc-His6 (A) or
pcDNA3.1-PE171-233-Myc-His6 (D-F) 48 hours prior to
fixation and processing. (A) Cells transfected with
pcDNA3.1-PE17-Myc-His6 were stained with DAPI (nucleus, blue), BODIPY
(lipid droplets, green), and probed with anti-myc (PE17;red). Images were captured by a confocal microscope and are
representative of two independent experiments. Scale bar = 5 µm. B)
Magnified view of the region of interest shown by the boxed in area in
A. Scale bar = 20 µm. C) Schematic cartoon showing the domain structure
of full-length PE17 and the truncated form, PE171-233.D-F) AECs were transfected with
pcDNA3.1-PE171-233-Myc-His6 48 hours prior to fixation
and processing. Cells were stained with DAPI (nucleus, blue)
and probed with anti-myc (PE171-233, red) and (D) BODIPY
(lipid droplets, green), (E) anti-Gpp130 (Golgi stack, green) or (D)
anti-COXIV (mitochondria, green). Insets show magnified views of areas
indicated by boxes. Scale bar = 5 µm.
FIGURE 6. PE17 inhibits eukaryotic cell growth and binds yeast
lipid droplets. (A) Yeast strains modified to induce GALpromoters with β-estradiol (Methods) and harboring either vector
controls or (B) pYES-PE17 or (C) pYES-PE17-mRuby2 were grown to
saturation in selective medium, diluted to OD600 = 1.0
in sterile water, then serially diluted 10-fold. 5 µl of each dilution
were spotted to CSM plates lacking uracil either lacking or containing 1
µM β-estradiol. Plates were incubated for 48h at 30°C and imaged. (D)
Saturated cultures from (C) were subcultured to 5 ml fresh medium either
lacking or containing 1 µM β-estradiol and outgrown for 6h at 30°C.
Total cellular proteins were extracted from equivalent cell densities as
in (von der Haar, 2007), separated via SDS-PAGE, and visualized via
immunoblot. Membrane was cut between the 25 and 37 kDa molecular weight
markers to blot for both PE17-mRuby2 (anti-Xpress, top) and Sec17p
(bottom). (E) Yeast strain harboring both GFP-Erg6 and the
galactose-inducible pYES-PE17-mRuby2 was grown to saturation in
selective medium at 30°C, subcultured to fresh medium containing 1 µM
β-estradiol to induce PE17-mRuby2 expression, outgrown for 6h at 30°C,
then visualized. Scale bar = 5 µm. (F) Yeast strains either lacking (3∆dga1∆ ) or producing lipid droplets in the presence of galactose
(3∆ GAL-DGA1 ) and harboring GFP-Erg6 and the galactose-inducible
pYES-PE17-mRuby2 plasmids were grown to saturation in selective medium
containing 2% glucose. Strains were subcultured to fresh selective
medium containing either 2% glucose or 2% galactose as a carbon
source. Cultures were grown for 18h at 30°C and cells were visualized
via epifluorescent microscopy. Scale bar = 5 µm. (G) Lipid droplets were
purified (Materials and Methods) from yeast strains harboring GFP-Erg6
and either vector control or PE17-mRuby2 plasmids. 15 µl from each lipid
droplet fraction were quickly mixed into 15 µl molten 0.6% agarose in
20 mM HEPES, 100 mM KCl, 2 mM MgCl2, pH = 7.4. 15 µl of
this mixture was mounted to a slide, allowed to solidify for 1 min, and
visualized via epifluorescent microscopy. Scale bar = 5 µm.