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SUPPLEMENTAL TABLE 1
Genetic analysis of glo-3(-) transheterozygotes
% of embryos that
lacked birefringent
granules in
intestinal cells
% of embryos with
1-10 enlarged
birefringent
granules in
intestinal cells
% of embryos
with 11-30
birefringent
granules in
intestinal cells
n
100
0
0
77
100
0
0
107
glo-3(kx90)
47
53
0
115
glo-3(zu446)
54
46
0
125
0
12
88
91
0
25
75
92
34
67
0
64
43
0
60
52
Genotype a
Class I allelesb
glo-3(kx91)
glo-3(kx94)
Class II alleles
b,c
Class III alleles
b
glo-3(kx1)
glo-3(kx38)
Class I x Class IId
glo-3(kx91) x glo-3(kx90)
glo-3(kx91) x glo-3(zu446)
57
glo-3(kx94) x glo-3(kx90)
21
79
0
glo-3(kx94) x glo-3(zu446)
51
49
0
61
13
31
56
54
54
Class II x Class III
d
glo-3(kx1) x glo-3(kx90)
glo-3(kx1) x glo-3(zu446)
4
20
76
glo-3(kx38) x glo-3(kx90)
3
48
49
58
glo-3(kx38) x glo-3(zu446)
17
51
32
53
All strains were grown at 22˚C. Pretzel stage embryos were analyzed using polarization
microscopy and scored for the presence, morphology, and number of birefringent granules
in the intestine.
a
Wild type embryos contain >100 birefringent granules in intestinal cells (Table 2).
b
Data for Class I, Class II, and Class III alleles are from Table 1.
c
Class II strains that contained the fer-1(hc13ts) mutation did not display an altered Glo
phenotype
d
Males containing a class I or class III allele were mated into class II allele containing
hermaphrodites. The class II hermaphrodites were self-sterile due to the presence of fer-
1(hc13ts) and being raised from embryogenesis to adulthood at 25oC. The resulting
progeny of these crosses are predicted to produce 50% males which are glo-3(class II
allele)/0 and should exhibit the class II phenotype and 50% hermaphrodites which are glo3(class I or class III allele)/glo-3(class II allele).
SUPPLEMENTAL TABLE 2
Colocalization of vital stains and autofluorescence in glo-3(-) adults
Genotype
Lysotracker Reda
Wild type
glo-3(kx94)
glo-3(kx90)
Nile Redb
Wild type
glo-3(kx94)
glo-3(kx90)
BODIPY
Wild type
glo-3(kx94)
glo-3(kx90)
TRITC-Dextran
Wild type
glo-3(kx94)
glo-3(kx90)
TRITC-BSA
Wild type
glo-3(kx94)
glo-3(kx90)
% of compartments displaying
specified fluorescence in anterior
intestinal cells
Autofluorescence Vital stain
only
only
Both
n
0
0
100
484
0
9
91
137
0
0
100
66
31
20
49
195
0
0
100
281
100
0
0
9
3
0
97
232
2
21
77
126
0
0
100
217
0
69
31
93
% of compartments displaying
specified fluorescence in posterior
intestinal cells
Autofluorescence
Vital stain
only
only
Both
n
0
2
1
0
4
4
100
94
95
365
430
242
0
33
47
0
5
1
100
62
52
54
239
249
0
100
100
0
0
0
100
0
0
297
175
45
1
31
1
0
0
8
99
69
91
279
98
242
0
11
1
3
4
12
97
85
87
226
223
130
All strains were grown at 22˚C. Individual young adults were stained and analyzed using
fluorescence microscopy with fluorescein isothiocyanate (FITC) and Rhodamine filters.
Images of anterior and posterior intestinal cells were taken with both filters and analyzed for
the colocalization of distinct puncta. When signals colocalized they were scored as Both.
Lysotracker Red, Nile Red, TRITC-Dextran, and TRITC-BSA staining was scored in
Rhodamine channel and autofluorescence was scored in the FITC channel. BODIPY staining
was scored in FITC channel and autofluorescence was scored in the Rhodamine channel. Since
gut granule autofluorescence is visible in both filters, images of vital staining were taken with
exposure times that did not reveal autofluorescence in unstained animals. Anterior cells of glo3(kx94) were not analyzed due the lack of autofluorescent organelles. n = number of puncta
scored for colocalization.
a
Lysotracker Red stained organelles in glo-3(-) animals were approximately 4-fold
dimmer than wild type.
b
Nile Red stained organelles in glo-3(-) animals were approximately 10-fold dimmer
than wild type.
SUPPLEMENTAL TABLE 3
Acidified and PGP-2 marked compartments in glo-3(-) adults
Genotype
% of animals with specified
number of stained compartments
in anterior intestinal cells
Very
Low Medium High
Low
n
% of animals with specified
number of stained compartments
in posterior intestinal cells
Very
Low Medium High
Low
n
Acridine Orange
Wild type
0
0
0
100 23
0
0
0
100
30
glo-3(kx94)
100
0
0
0
12
8
92
0
0
12
glo-3(kx90)
17
83
0
0
18
0
89
11
0
18
α-PGP-2 antibodies
Wild type
0
0
0
100 40
0
0
0
100
17
glo-3(kx94)
61
36
3
0
28
5
74
21
0
19
glo-3(kx90)
0
96
4
0
27
7
67
26
0
15
All strains were grown at 22˚C. Young adults were fed Acridine Orange or fixed and immunostained
with anti-PGP-2 antibodies. Individual Acridine Orange stained young adults were analyzed using a
rhodamine filter and scored for the number of stained organelles within anterior and posterior
intestinal cells. Anti-PGP-2 stained animals were similarly scored. Animals were scored as Very Low
when 0-5 fluorescent compartments were present, as Low when 6-100 fluorescent compartments were
present, as Medium when 101-200 fluorescent compartments were present, and as High when >200
fluorescent compartments were present. n = number of animals scored.
SUPPLEMENTAL FIGURE LEGENDS:
SUPPLEMENTAL FIGURE 1. Analysis of gut granules in glo-3(-) adults. (A-F) Wildtype intestinal cells contained many organelles that brightly stain with markers for fat
(B), acidity (D), and terminal endocytic activity (F). glo-3(-) adults lacked BODIPY
staining of fat (H), and contained dramatically reduced numbers of acidified (J) and
terminal endocytic compartments (L). In A-L the intestinal lumen is marked with a black
arrow. Posterior intestinal cells are shown in all panels.
SUPPLEMENTAL FIGURE 2. Sudan Black staining is unaltered in glo mutants. (A-D)
Wild-type and glo(-) young adults were fixed and stained with Sudan Black. Posterior
intestinal cells are shown in all panels.
SUPPLEMENTAL FIGURE 3. smg-1(r861) partially suppresses the Glo phenotype
exhibited by some glo-3(-) alleles. The intestinal cells of wild-type (A) and smg-1(r861)
(B) pretzel stage embryos displayed many birefringent organelles and did not mislocalize
birefringent material into the intestinal lumen. glo-3(-) embryos mislocalized
birefringent material into the intestinal lumen (arrows in C, E, G, I) and lacked (C) or
rarely contained birefringent material within their intestinal cells (E, G, I). smg-1(r861);
glo-3(kx94) embryos exhibited the same phenotype as glo-3(kx94) (D). In contrast, smg1(r861) dramatically altered the phenotype of three other glo-3 (-) alleles (F, H, J). These
smg-1(r861); glo-3(-) embryos did not mislocalize birefringent material into the intestinal
lumen and contained a significant number of enlarged birefringent granules within their
intestinal cells (F, H, J). Intestinal cells are located between the black arrowheads. Black
arrows denote the location of the intestinal lumen.