Live-cell biosensor polypeptides and methods of use
View Patent ↗Methods and compositions for determining the duration of a cell cycle phase of a mammalian cell, as well as identification of the cell cycle stage of fixed mammalian cells, are provided. In practicing the subject methods, at least one biosensor polypeptide that monitors a cell-cycle phase in a mammalian cell, such as mitosis, G1, S, or G2 phase, is used to determine the duration of a cell-cycle phase of a mammalian cell. Also provided are methods for identifying an agent (e.g., a gene product or small molecule compound) that modulates the duration of a cell-cycle phase of a mammalian cell, as well as kits and systems for practicing the subject methods.
1. A method of monitoring a cell-cycle phase of a mammalian cell, the method comprising:
culturing a mammalian cell comprising
a mitosis biosensor polypeptide (MBP) comprising a plasma membrane targeting domain, a nuclear localization signal and a fluorescent label,
a G1 phase biosensor polypeptide (G1BP) comprising a fluorescent label, and
a S phase biosensor polypeptide (SBP) comprising a fluorescent label,
under conditions suitable for proliferation of the mammalian cell, wherein each of the fluorescent labels of the G1BP, SBP, and MBP emit different fluorescence emission spectra; and
detecting a MBP fluorescence pattern and at least one of
a) G1BP fluorescence pattern established by a nuclear localized fluorescence and a cytoplasmic localized fluorescence; and
b) SBP fluorescence pattern established by formation of fluorescence puncta in a nuclear region of the mammalian cell and disappearance of fluorescence,
wherein nuclear envelope breakdown (NEB) is characterized by MBP fluorescence localized to a plasma membrane, indicating a mitosis phase of the cell cycle, and nuclear envelope reformation (NER) is characterized by MBP fluorescence localized to a nucleus, indicating interphase of the cell cycle.
2. The method according to claim 1 , wherein prior to said detecting the cells are fixed.
3. The method according to claim 1 , wherein a G1BP fluorescence pattern established by nuclear localized fluorescence is indicative of G1 phase of a cell cycle.
4. The method according to claim 1 , wherein a G1BP fluorescence pattern established by cytoplasmic localized fluorescence is indicative of S or G2 phase of a cell cycle.
5. The method according to claim 1 , wherein a SBP fluorescence pattern established by formation of fluorescence puncta in a nuclear region of the mammalian cell is indicative of S phase of a cell cycle.
6. The method according to claim 1 , wherein detection of the MBP fluorescent pattern is by total internal reflection fluorescence (TIRF) microscopy.
7. The method according to claim 1 , wherein cell cycle phase duration is determined in a plurality of cells and in parallel.
8. A method for determining duration of a cell-cycle phase of a mammalian cell, the method comprising:
culturing a mammalian cell comprising
a mitosis biosensor polypeptide (MBP) comprising a plasma membrane targeting domain, a nuclear localization signal and a fluorescent label,
a G1 phase biosensor polypeptide (G1BP) comprising a fluorescent label and
a S phase biosensor polypeptide (SBP) comprising a fluorescent label
under conditions suitable for proliferation of the mammalian cell, wherein each of the fluorescent labels of the G1BP, SBP, and MBP emit different fluorescence emission spectra; and
determining a time interval between at least two of
a) NEB,
b) NER,
c) formation of fluorescence puncta in a nuclear region of the mammalian cell,
d) disappearance of fluorescence puncta in a nuclear region of the mammalian cell,
e) appearance of fluorescence in a nuclear region of the mammalian cell, and
f) translocation of fluorescence from a nuclear region to a cytoplasmic region of the mammalian cell,
wherein a), b), c), d), e), and f) are established by detection of at least one of a MBP fluorescence pattern, a G1BP fluorescence pattern, or a SBP fluorescence pattern
wherein the time interval between NEB and NER is indicative of duration of a mitosis phase of the cell cycle and the time interval between NER and NEB is indicative of duration of interphase of the cell cycle.
9. The method according to claim 8 , further comprising determining a time interval between at least two of NEB, prometaphase, metaphase, anaphase, cytokinesis, and NER, wherein NEB and NER are established by detection of a MBP fluorescence pattern and prometaphase, metaphase, anaphase, cytokinesis are established by detection of a condensed chromatin associated M BP fluorescence pattern.
10. The method according to claim 8 , wherein the time interval is between NER and formation of fluorescence puncta in a nuclear region of the mammalian cell, which time interval is indicative of duration of G1 phase of a cell cycle.
11. The method according to claim 8 , wherein the time interval is between formation of fluorescence puncta in a nuclear region of the mammalian cell and disappearance of fluorescence puncta in the nuclear region of the mammalian cell, which time interval is indicative of duration of S phase of a cell cycle.
12. The method according to claim 8 , wherein the time interval is between disappearance of fluorescence puncta in a nuclear region of the mammalian cell and NEB, which time interval is indicative of duration of G2 phase of a cell cycle.
13. The method according to claim 8 , wherein the time interval is between NER and translocation of fluorescence from a nuclear region to a cytoplasmic region of the mammalian cell, which time interval is indicative of duration of G1 phase of a cell cycle.
14. The method according to claim 8 , wherein the time interval is between translocation of fluorescence from a nuclear region to a cytoplasmic region of the mammalian cell and disappearance of fluorescence puncta in a nuclear region of the mammalian cell, which time interval is indicative of duration of S phase of a cell cycle.
15. The method according to claim 8 , wherein the time interval is between appearance of fluorescence in a nuclear region of the mammalian cell and translocation of fluorescence from a nuclear region to a cytoplasmic region of the mammalian cell, which time interval is indicative of duration of G1 phase of a cell cycle.
16. The method according to claim 8 , wherein detection of the MBP fluorescent pattern is by total internal reflection fluorescence (TIRF) microscopy.
17. The method according to claim 8 , wherein cell cycle phase duration is determined in a plurality of cells and in parallel.
18. The method according to claim 8 , wherein said culturing is performed in the presence of a candidate agent, wherein an increase or decrease in the time interval as compared to a time interval in the absence of a candidate agent indicates the candidate agent modulates duration of a phase of a cell cycle of the mammalian cell.