Thesis Progress: Week 11 and 12
I did an initial test of fluorescence
To see the incredible motion of the cells and the lamellipodia and filopodia is thrilling.
The film focuses on a cell with massive lamellipodia as it is undergoing mitosis.
After a few unsuccessful attempts, the transfection is successful.
I used the Fluorescence Ubiquitination Cell Cycle Indicator developed by Miyawaki and colleagues that takes advantage of biphasic cycling of geminin and Cdt1 to tie different fluorescent proteins to cell cycle.
Modification of protocol:
The protocol called for a specific index of viral particles per cell.
Approx 40ppc at 1ml labeling volume for a 1×10^8 of virus.
The breakthrough, was actually increasing the viral particles to 100 and halving the labeling volume to .5ml.
Additionally media contains phenol red, a salt that creates a ph marker.
However it sometimes interferes with fluorescence and can actually cancel out any fluorescence.
Incubation for 25 hours in 37C w 5%
Specifically by using a baculovirus for transfection I modified Swiss 3T3 cells to the following scheme.
[In the G1 phase of the cell cycle, geminin is broken down and only Cdt1 tagged with RFP may be visualized, thus identifying cells in the G1 phase with red fluorescent nuclei.
In the S, G2, and M phases, however, Cdt1 is degraded and only geminin tagged with GFP remains, thus identifying cells in these phases with green fluorescent nuclei.
During the G1/S transition, as Cdt1 levels decrease and geminin levels increase, both proteins are present in the cells, allowing GFP and RFP fluorescence to be observed—when green and red images are overlaid, the cells appear with yellow fluorescent nuclei.
This dynamic color change from red-to-yellow-to-green represents the progression through cell cycle and division.]
Next steps:
I am in the process of finalizing the timelapse of the cells, and anticipating to have something in the next few days.
I have gotten very interested in oscillators and synchronization.
I had a brief meeting with Natalie Jerimejenko and followed her recommendation to become more familiar with the work of American mathematician, Stephen Strogatz and principal researcher at Yahoo, Duncan Watts.
There are some differential equations the tackle but the topic is fascinating and I just acquired both Sync and Nonlinear Dynamics and Chaos.
I wonder if there is any relation between the frequency and period x/1 of 3T3 and other mass and directed phenomena.
Will the cells move towards synchrony and what will happen when they reach confluency.
I think this specific line is semi transformed, although I believe I let them become 100% confluent during the contamination period, so perhaps now that they have fully mutated they will keep growing expressing colors in ever increasing density.
My Ofx program is complete and is capturing averages of colors per thresholded color value.
I believe this will be at least a basic way of analyzing the populations of cell per particular cell phase.
There is another image analysis program targeted for these types of application that is pretty interesting called ImageJ. Unfortunately for me, it is written in Java. Perhaps there is a GUI that provides sufficient functionality. I will explore.
Ultimately the next step is to express cell cycles as cellular events and into macro/physical events.
N.B. A very quick overview of the cell cycle phases:
Overall there are 2 main cell parts
Interphase and Mitosis (specifically in eukaryots)
Interphase is the period when the cell is actively growing and synthesizing RNA into amino acids and proteins, and duplicating its DNA/chromosomes
This period can be divided in G1, S, G2
G1 ( Gap1 ) phase {expressed in red} is RNA and protein synthesis.The cell is growing and putting everything in order for the next phase.
S ( Synthesis ) phase {expressed in yellow} is the cell duplicating its DNA material.
Each chromosome has now 2 sister chromatids.
G2 ( Gap2 ) phase {expressed in green) DNA synthesis is complete and the cell continues biosynthesis for components necessary for the following- Mitosis phase.
Mitosis is generally split into these stages:
prophase- [before] DNA material forms into chromosomes
prometoephase – the nucleus breaks apart
metaphase- [adjacent] Chromosomes move to the center of the nucleus
anaphase – [up] chromosomes move to opposite ends of the cell
telophase- [end] nuclear envolope transforms into 2
Once the chromatids have been separated, cytokinesis commences distributing all the other cell components to each sister cell.
There are now 2 sister cells and G1, S, G1, M repeats every 18 hours in Swiss 3T3 cells.
[In the G1 phase of the cell cycle, geminin is broken down and only Cdt1 tagged with RFP may be visualized, thus identifying cells in the G1 phase with red fluorescent nuclei.
In the S, G2, and M phases, however, Cdt1 is degraded and only geminin tagged with GFP remains, thus identifying cells in these phases with green fluorescent nuclei.
During the G1/S transition, as Cdt1 levels decrease and geminin levels increase, both proteins are present in the cells, allowing GFP and RFP fluorescence to be observed—when green and red images are overlaid, the cells appear with yellow fluorescent nuclei.
This dynamic color change from red-to-yellow-to-green represents the progression through cell cycle and division.]
