a
b
ASH1 mRNA expression and localization during the cell cycle
Tub1 protein
ASH1 mRNA
G1
S
G2
M
bud
mother
Tub1 protein
(Alexa 555)
ASH1 mRNA
(Quasar 670)
DIC
DAPI
MERGE
M(Ana)
M(Ana)
M(Ana)
G1
S
Tub1 IF
DIC
DAPI
ASH1 mRNA smFISH
Fig. 4 smFISH-IF for the cyclin ASH1 mRNA and the tubulin protein. (a) Schematic representation of ASH1
mRNA expression and localization during the cell cycle. Green dots represent ASH1 mRNA in the cytoplasm
and transcription sites in the nucleus, which are brighter than the cytoplasmic mRNAs. Tubulin 1 co-localizes
with the spindle pole body, which is duplicated during S phase. The bud emergence starts during S phase and
ends with the formation of the daughter cell. During anaphase, the microtubules stretch between the mother
and the daughter cell. At this time, the ASH1 mRNA is rapidly transcribed and it localizes at the bud tip. (b)
MERGE maximally projected image: ASH1 mRNA smFISH Quasar 670 (green), tubulin IF Alexa 555 (magenta),
and DAPI (blue) merged to a single-plane DIC image (gray). The cell cycle phase of few representative cells is
indicated on the image. Scale bar 10 μm (c); maximally projected channels from image shown in (b) are
individually displayed
CLN2 and ASH1 mRNA expression and localization during the cell cycle
Tub1 protein
ASH1 mRNA
CLN2 mRNA
G1
S
G2
M
bud
mother
Tub1 protein
(Alexa 488)
ASH1 mRNA
(Quasar 670)
CLN2 mRNA
(Quasar 570)
DIC
DAPI
MERGE
Early G1
G1
Late G1
S
G2
Mitosis (P) Mitosis (A) Mitosis (A)
Mitosis (T)
a
b
Fig. 5 smFISH-IF for the cyclin CLN2 and ASH1 mRNAs and the tubulin protein. (a) Schematic representation of
CLN2 and ASH1 mRNA expression and localization during the cell cycle. (b) MERGE maximally projected
image: ASH1 mRNA smFISH Quasar 670 (green), CLN2 mRNA smFISH Quasar 570 (yellow), tubulin IF Alexa
488 (magenta), and DAPI (blue) merged to a single-plane DIC image (gray). The cell cycle phase of few
representative is indicated on the top of the image (P prophase; A anaphase; T telophase). Scale bar 3 μm
62
Evelina Tutucci and Robert H. Singer
b
ASH1 mRNA expression and localization during the cell cycle
Tub1 protein
ASH1 mRNA
G1
S
G2
M
bud
mother
Tub1 protein
(Alexa 555)
ASH1 mRNA
(Quasar 670)
DIC
DAPI
MERGE
M(Ana)
M(Ana)
M(Ana)
G1
S
Tub1 IF
DIC
DAPI
ASH1 mRNA smFISH
Fig. 4 smFISH-IF for the cyclin ASH1 mRNA and the tubulin protein. (a) Schematic representation of ASH1
mRNA expression and localization during the cell cycle. Green dots represent ASH1 mRNA in the cytoplasm
and transcription sites in the nucleus, which are brighter than the cytoplasmic mRNAs. Tubulin 1 co-localizes
with the spindle pole body, which is duplicated during S phase. The bud emergence starts during S phase and
ends with the formation of the daughter cell. During anaphase, the microtubules stretch between the mother
and the daughter cell. At this time, the ASH1 mRNA is rapidly transcribed and it localizes at the bud tip. (b)
MERGE maximally projected image: ASH1 mRNA smFISH Quasar 670 (green), tubulin IF Alexa 555 (magenta),
and DAPI (blue) merged to a single-plane DIC image (gray). The cell cycle phase of few representative cells is
indicated on the image. Scale bar 10 μm (c); maximally projected channels from image shown in (b) are
individually displayed
CLN2 and ASH1 mRNA expression and localization during the cell cycle
Tub1 protein
ASH1 mRNA
CLN2 mRNA
G1
S
G2
M
bud
mother
Tub1 protein
(Alexa 488)
ASH1 mRNA
(Quasar 670)
CLN2 mRNA
(Quasar 570)
DIC
DAPI
MERGE
Early G1
G1
Late G1
S
G2
Mitosis (P) Mitosis (A) Mitosis (A)
Mitosis (T)
a
b
Fig. 5 smFISH-IF for the cyclin CLN2 and ASH1 mRNAs and the tubulin protein. (a) Schematic representation of
CLN2 and ASH1 mRNA expression and localization during the cell cycle. (b) MERGE maximally projected
image: ASH1 mRNA smFISH Quasar 670 (green), CLN2 mRNA smFISH Quasar 570 (yellow), tubulin IF Alexa
488 (magenta), and DAPI (blue) merged to a single-plane DIC image (gray). The cell cycle phase of few
representative is indicated on the top of the image (P prophase; A anaphase; T telophase). Scale bar 3 μm
62
Evelina Tutucci and Robert H. Singer