Rho Rho Rho Your Boat
This week I studied yet another protein thought to play a role in cell division: Cyk 4. In order to explain what we think Cyk 4 does, I first have to explain a little more about cell division. One of the things that the cell has to successfully do in order to divide is create a cleavage furrow in the proper place to pinch the cell into two equal half with a copy of DNA in each new half. In order to do this protein called Rho “paints” the cell in a band where it should divide. This band of Rho then recruits contractile proteins to form the cleavage furrow and start cytokinesis. Another protein called Ect 2 tells Rho the correct place to “paint” the cell. In order to do its job Ect 2 has to be transported. Cyk 4 may have a role in transporting Ect 2 allowing everything described above to happen.
To test this we again used a morphlino to “turn off” the gene that codes for the production of Ect 2 to see how this would effect cell division. We compared normal eggs that could make Ect 2 with the eggs we “turned off” the Ect 2 gene in. We injected both types of eggs with a probe which allowed us to see the amount of Rho in the cleavage furrow. We viewed the normal control eggs with the confocal microscope first after allowing. Below is one of videos we took of a control after it had undergone a few rounds of division.
In this video you can see the bands of Rho activity in the cleavage furrow as the cells divide. Then we looked at the cells without Ect 2 after a few rounds of division.
See You Next Week!
Thanks so much for reading. For all of you who made it to the end here’s a photo I took of Cape Perpetua this weekend and also a photo of a llama I met.
The protein I have been focusing on this week is called Mad 2. It is thought to be involved with a checkpoint in cell division that prevents the cell from going through division until all the chromosomes are properly attached to the meiotic spindle. Then they can be dragged apart, leaving each new daughter cell with a copy of each chromosome. If something goes wrong with this process chromosomes may be left behind or both copies will end up attached to only one end of the spindle complex and one of the daughter cells will end up with both copies of the chromosome. Mad 2 may be a protein that serves as a “wait for me” signal that unattached chromosomes release to keep the cell from dividing without them. This week, we “turned off” the genes that code for Mad 2 with a morpholino solution we injected into starfish oocytes (just like I talked about with the Anillin in the week 3 blog post). We then viewed them with a Hoechst dye which highlights DNA under certain light conditions. Below is a photo I took which shows the nuclei of a blastula (fertilized egg that has divided until it is a hollow ball of cells).
We also injected some of the oocytes with two fluorescent probes to allow us to see microtubules and DNA under the confocal microscope. This allowed us to look for abnormalities in the cells deprived of Mad 2. Below are some examples of the type of videos we are able to make.
In this video you can watch the cell undergo second meiosis. The chromosomes are seen in blue and the microtubules in yellow. The cell halves its DNA and then pushes it out into a polar body.
In this video you see the cell's first division. First the microtubules deposited by the sperm pulls the oocyte's pronucleus towards its own. Then the DNA in the nucleus is wound into chromosomes and pulled apart by the spindle fibers. At the very end you can see the cell start to cleave in two.
The babies are still growing along, but I haven’t had much time to look at them. Claire, my fellow intern, took some wonderful photos of our baby phoronids though. Their little tentacles are getting so long. If you want to know more about the development of our little larva Claire has a great blog on their development which she posted last week.
See You Next Week!
Thanks so much for reading. For all of you who made it to the end: I give you a photo I took last weekend while car camping at Mount Hood.
Lights, Camera, Action!
This week I have done a lot of filming embryos and watching their development. We spent the early part of the week preparing by injecting starfish eggs with the morphlino solution that would effectively “turn off” certain genes we are studying. By seeing how the cell reacts when the gene cannot be expressed, we learn about the function that protein has in development. Later in the week we matured and fertilized these eggs so they would start to divide. We filmed them overnight with a time lapse camera attached to a microscope and got to see how they did in the morning. Below is a video (please excuse the poor quality, I had to take a video of the screen because of the large file size that the microscope camera requires). I took of some cells which had been injected with a gene knockdown solution to turn off the expression of a specific gene. The blue cells at the bottom which eventually float off screen (a very frustrating problem I keep encountering) are normal cells that we stained as a control. Notice that the yellowish cells that have been injected divide later than the control, divide unevenly, and never reach the blastocyst (hollow ball of cells) stage like the controls. Clearly the cells are having trouble without being able to express the gene that I turned off.
Our little larvae are growing so fast! This week Claire put our Phoronids under the dissecting scope (a microscope that allows us to view organisms swimming in water) and discovered they had grown little squid-like arms! Our Patiria are getting HUGE. I can see them clearly without a microscope now. Also, our urchin larva now look like little spaceships. Watching these guys grow is really fun.
Thanks For Reading!
Just for you few who read all the way to the end, a bonus picture I took a few days ago in Bandon, Oregon. This place is so beautiful!
“Do you know how a cell divides?… The answer is no. All biologists should say no”
-George von Dassow
Cell division is exceedingly complicated. I have spent the last week trying to wrap my head around it. The more I learn, the more I realize I don’t know. Apparently, I am not alone in this feeling. George von Dassow has been studying this very subject for years and still has questions about how it all works. Since I am jumping into a small part of his research on the subject for my REU, I will attempt to explain my little bit here.
Cell division involves many, many proteins which work together to form a contractile ring which pinches the cell into two parts each containing a single copy of the original cell’s duplicated DNA. Many of these proteins’ role in this process is not clearly understood. I will be focusing on the protein Anillin and its role in cell division for my project. This protein may have a regulating effect on cell division, keeping the cell from becoming too excitable while forming the contractile ring. To study this protein I will be using something called gene knockdown. Gene knockdown is a process by which we can artificially turn off specific genes in the cell by blocking the RNA instructions to make that specific protein. Using the micromanipulator I practiced with last week, I will be injecting starfish oocytes with morpholino antisense oligonucleotides (a segment of specialized RNA that will attach to the gene for Anillin on the cells mRNA), hopefully blocking the cell from being able to use those mRNA instructions to make the protein Anillin.
Then, I will fertilize these cells and see if the gene knockdown will cause anything abnormal about their division. From observing what happens to these cells I may be able to draw conclusions on the roll of Anillin during cell division.
Baby Watch 2017
The babies are growing up so fast! I have been diligently changing their tank watering and feeding them a healthy diet of algae. As promised, I have baby photos.
I also sacrificed and stained some Patiria larva to view them with the confocal microscope. The confocal is a powerful tool that creates beautiful and detailed images. By staining we are able to only view particular structures in the cells. We stained larva for nuclei (seen in blue) and actin filaments (seen in green). You can see a clear outline of the larva in blue because the cells on the outer surface are tightly packed together into a ciliated band.
We also used the confocal to view the image in several “slices” which I used to put together the video below.
See You Next Week!
Thanks so much for reading.
Week 2 at OIMB
The last two weeks have been FULL! I can hardly decide what I want to talk about. One of my favorite experiences was going tide pooling at Cape Argo. It was awesome to see so many alien-looking creatures everywhere. We were attempting to find sea cucumbers for another students research. These little guys can be tricky because they hide in rocks and inflate themselves making it impossible to pull them out. While we were looking for these elusive invertebrates under various rocks, we came across an octopus! I was a little nervous it would bite me (right before handing it to me someone told me that one had bitten a student the quarter before and his hand swelled up), but luckily both me and the octopus parted ways unscathed
My name is Nicolle Koontz and I am from Grass Valley, California where I attend Sierra College. I am lucky enough to be working in George von Dassow's lab doing research on the cellular biology of starfish oocytes during mitosis. I am an information addict. I have to know how things work, how we know how those things work, and why they work the way they do. These sorts of questions have led me to major in Biology. As an REU intern I look forward to working in a state of the art lab with mentors and grad students who share in my joy of discovery while also building my skill set. I am so excited to dive into a project that can contribute to our collective scientific understanding of the world.