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 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.
------------
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
No comments:
Post a Comment