Visualizing Spatiotemporal Dynamics of

Multicellular Cell-Cycle Progression

 Cell cycle
The cell cycle, is the series of events that take place in a cell leading to
duplication of its DNA (DNA replication) and division of cytoplasm and
organelles to produce two daughter cells.
 ubiquitination
1)The addition of ubiquitin to a substrate protein is called ubiquitination
2)Ubiquitination requires three types of enzyme:
• ubiquitin-activating enzymes(E1)
• ubiquitin-conjugating enzymes( E2)
• ubiquitin ligases ( E3)
 FUCCI
• Fluorescent Ubiquitination-based Cell Cycle Indicator

• FUCCI utilizes replication licensing factors Cdt1 and Geminin, uses the
reciprocal periodicity of cdt1 and geminin expression.

• A fusion protein of a following fragment are prepared

1)Cdt1 (amino acids 30-120) fragment with the fluorescent protein
monomeric Kusabira-Orange 2 (mKO2): visualise G1 phase.
2) Geminin (amino acids 1-110 ) fragment with the fluorescent protein
monomeric Azami-Green 1 (mAG1) :visualizes the S,G2 and M phase.

• FUCCI utilizes the rapid degradation of the replication licensing factors

mediated by the ubiquitin proteasome system .
• fused red- and green-emitting fluorescent proteins to E3 ligase substrates,
Cdt1 and Geminin, to develop dual-color fluorescent probes that indicate
whether individual live cells are in G1 phase or S/G2/M phases. .

• mKO2 was fused to full-length human Cdt1 (hCdt1)

• When the chimeric protein was expressed in HeLa cells under the control
of the ubiquitous CMV promoter, red fluorescence was observed.

• first 48hr after transfection, observe sudden disappearance of red

fluorescence, suggesting that mKO2-hCdt1 protein was being degraded by
the SCFSkp2 complex at the onset of Sphase.

• after 48 more hour the transfected cells failed to proceed to mitosis,

whereas nontransfected cells divide.
• To overcome the cell-cycle arrest, numerous hCdt1 deletion mutants were
constructed .

• And fused to mKO2 then evaluated for cell cycle-dependent red fluorescence
in the nucleus.

• The Cymotif(amino acids 68 – 70), which binds to the SCFSkp2 E3 ligase , was
required .

• N-terminus of hCdt1 (amino acids 1–10) binds to a different E3 ligase (Cul4) ,

removal of this region .

• Cdt1 degradation by the SCFSkp2 complex has been shown to be

independent of its binding to Geminin ,removed theGeminin-binding region.

• The resulting truncated hCdt1protein(aminoacids30–120) is sufficient for

marking cells in G1 phase [mKO2-hCdt1(30/12)
 Fucci plasmids

Lentiviral construction

the cells

Selection

Imaging
 Fucci plasmids

Lentiviral construction

Transducing the
cells

Selection

Imaging
 Fucci plasmids

Lentiviral construction

Transducing the
cells

Selection

Imaging
 MCF10A
MCF-10A is an
adherent,
immortalized,
non-transformed human mammary epithelial cell line .
• These cells were originally derived from a 36-year-old
patient with fibrocystic changes .
MCF-10A cells lack tumorigenicity in nude mice
lack of anchorage-independent growth,
dependence on growth factors and hormones for
proliferation and survival.
 Growth Medium
• DMEM:F12
• 5% horse serum
• 0.5 μg/ml hydrocortisone ,
• 20 ng/mlhEGF ,
• 10 μg/ml insulin ,
• 100ng/ml cholera toxin , and
Passage time:
-MCF-10A cells should be passaged every 3-4 days.
- Cells should never be grown over 80% confluency.
• Fucci plasmids

Bacterial transformation

plasmid isolation

Agarose gel elctrophoresis