Yeast study reveals novel regulators of cell division

6:29:00 PM

Emily Pugach, 5th year PhD candidate in Molecular, Cellular and Developmental Biology

Ever think back to the time when you were just a single cell? Probably not. But consider the fact that your adult body is composed of upwards of 37 trillion cells!  While we may take for granted this astounding accomplishment, it’s no small feat. How does a human embryo go from one cell to 37 trillion cells? Well, simple as it may sound, that first cell makes copies of itself and each of those new cells follows in turn to build a living, breathing human being.  

The propagation of one cell to multiple cells is accomplished through cell division, a part of the cell cycle known as mitosis. Successful mitosis requires much more than cell division: first, it requires extremely careful multiplication of both cellular material and genetic information encoded in DNA. This ensures that each cell in the body ends up with the accurate and identical genetic material essential for an organism to propagate and survive. Although a seemingly straightforward process, precisely dividing cellular contents, especially each of the duplicated chromosomes in a cell’s nucleus, requires careful regulation.

A cell duplicates its contents, including its DNA (orange X’s) and centrosomes
 (red boxes) and divides them equally between two daughter cells.
DNA is partitioned using a spindle apparatus (red boxes and blue lines).
 
Source: Wikimedia.org
 In cases where chromosomes are not divided appropriately, the products of cell division, known as the daughter cells, will contain inappropriate genetic information. For example, one daughter cell may contain multiple copies of a particular chromosome while another may contain no copies of the same chromosome. As you might expect, inheriting the wrong number of chromosomes can be a big problem for a cell, and when those damaged cells divide it can be an even bigger problem for the entire organism. 

In many cases of DNA segregation errors, a cell will “sense” a particular error and stop its division process immediately. But imagine a cell like this bypasses the quality control process. Now consider what could go wrong if this damaged cell makes more copies of its imperfect DNA and passes them on to its own daughter cells.  This hypothetical scenario happens all too often. Many cancer cells arise from these types of cell division defects. Jennifer Avena, a former graduate student in the department of Molecular, Cellular and Developmental Biology, sought to understand exactly how a cell knows when it is safe to proceed with division.

When she undertook this project, Jennifer had a generous body of research to draw from. It is known, for example, that when a cell divides, its duplicated DNA is separated to its daughter cells on a set of cellular machinery known as the centrosome and spindle. In the yeast cells that Jennifer studied, an analogous structure, the spindle pole body, performs the same role as the centrosome; organizing the chromosomes for separation. One centrosome or spindle pole body will eventually be allocated to each daughter cell, so this apparatus must also be duplicated before a cell divides into two. How does a cell carefully regulate the replication and partition of its DNA, spindle pole body, and centrosomes? Answering this question became the crux of Jennifer’s research.
 
A yeast cell replicates its chromosomes (DNA) and undergoes
either normal or abnormal DNA segregation facilitated by the spindle
pole body and associated cellular machinery. Abnormal DNA segregation,
as in the case of the purple chromosome 1, can lead to cancer. 
In fact, while the general players that regulate cell division and DNA replication have been identified, exactly how these players execute their regulation is not known. Imagine seeing a traffic signal for the first time and appreciating the fact that traffic was regulated but not understanding the message that each colorful signal conveyed. In the case of regulating cell division, Jennifer knew from other studies that a type of protein called a kinase acts like a traffic light. The kinase, known as Cdk1, exerts its effect by phosphorylating other molecules- a process in which it attaches small chemical modifications onto its substrates. 

These modifications, or phosphorylation marks, are attached to thousands of molecules in a cell to modify their activities or denote to another molecule a change in status. For Cdk1, Jennifer identified specific sites on a particular target, Sfi1, as being critical indicators to a cell that division should proceed. In essence, when Sfi1 was phosphorylated, or flagged, by Cdk1, other components of a cell’s machinery were notified that all was normal and to proceed dividing DNA and other cellular components.

In order to demonstrate the importance of Cdk1 regulation of Sfi1, Jennifer used a clever trick. She mutated the Sfi1 molecule so that the sites normally flagged by phosphorylation were no longer able to be phosphorylated by Cdk1. In this way, she created a broken traffic light, essentially stuck on green. As she expected, cells that contained this mutated version of Sfi1 often proceeded through cell division without properly segregating their components. They even made multiple copies of their spindle pole bodies.

Jennifer was able to observe this outcome directly in the cells she studied using a technique called electron microscopy (EM). EM allows for ultra high magnification and resolution of tiny subcellular structures like spindle pole bodies using accelerated electrons as a source of illumination. CU has a state of the art electron microscope that Jennifer and her colleagues were able to take advantage of.

Jennifer soon realized that she was looking at something new and significant under the electron microscope: “The best word to describe our finding of reduplicated spindle pole bodies in multiple Sfi1 mutants would be ‘exciting.’  This phenotype had only been seen…in one other budding yeast mutant, one in which several [known regulators of cell division] are deleted.  Our findings led us to the model that premature [spindle pole] duplication is blocked by phosphorylation of Sfi1.” In essence, the cross-talk between Cdk1 and Sfi1 serves as a kind of master regulator of spindle pole duplication during cell division.

Many of the cells Jennifer observed had vast defects beyond spindle pole body reduplication; some could barely grow at all. This was not surprising given the cell’s mistaken “decision” to create extraneous machinery for separating cellular components. It was clear to Jennifer and her collaborators that Cdk1 regulation of Sfi1 is an absolutely essential step in proper cell division. Miscommunication between these two players has dire consequences on a cell’s ability to undergo appropriate replication.

As pointed out above, the cells used for Jennifer’s studies were yeast cells, like those found in bread and beer. However, the components she characterized as playing critical roles in regulating cell division are found in many species, even humans. Working with yeast cells made Jennifer’s task of testing her hypothesis about the importance of Sfi1 and Cdk1 much easier than doing similar studies in human cells and the results are likely still applicable to cell division in humans. Although it is known that Sfi1 and Cdk1 exist in human cells, Jennifer is looking forward to assessing whether the role of these proteins in regulating spindle pole duplication is also conserved in humans. She is also curious what other molecules they may be interacting with and the implications of these interactions.

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Jennifer defended her thesis in 2014 and is currently pursuing post-doctoral research at Vanderbilt University in the laboratory of Kathy Gould. Jennifer continues to study cell division in yeast.

Jennifer and her collaborators published the entirety of their findings in PLOS Genetics in October, 2014. You can read their detailed report here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207612/pdf/pgen.1004666.pdf

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