Biology 355

Project 5 Centrioles

Spring 2015

Many of you became intensely interested in cell division and regulators of cell division because of
your interest in human disease, in particular cancer. While I suspect many of you are reasonably
well versed in cyclins and cyclin dependent kinases (CDKs), there are lots of other regulators
necessary for cell division to occur with appropriate constraints. Centrioles are key elements in
the story of how cells divide their genetic material. The paper we will read is about regulating
centriole duplication. That regulation requires you to know about how proteins themselves get
regulated post-translationally.
Learning objectives of this project:
- Solidify your understanding of why immunocytochemistry, RNAi, and Co-IP are used and
how to interpret new experiments that involve these methods. Apply knowledge of protein
tags to GFP-fusions to track a protein of interest.
- Understand the role of centrioles in cell division AND understand how they themselves are
regulated.
- Understand ubiquitin and how regulating its addition can help control protein availability
and cellular functions.
- Consider paper supplements. Why bother why not just publish more papers? What
information is reserved for the supplements, how do you find them?
- Consider open-access journals. Why publish there, what do they offer that peer reviewed
journals do not (and vice versa)?
Part 1
A) PREPARE (in advance)
1 Use your textbook.
a) Look up what centrioles do. Dont just rely on your memory look it up. What are they composed of,
how are they made, how and when are they duplicated, and what do they do? 6th edition of Karp

chapter 9, section 9.3 on microtubules.

b) Look up what ubiquitin is. How does it get added onto proteins and what happens to proteins that get
ubiquitinated? 6th edition of Karp, chapter 12, section 12.7.
2 Read a science digest article Birth Control for Centrioles) Science Daily
http://www.sciencedaily.com/releases/2009/01/090126100640.htm
Note that this article is older and discusses the process of regulating centriole duplication from a fruit fly
perspective. Our paper will be more recent and will focus on the C. elegans (worm) equivalent story.
Guiding questions:
1)
2)
3)
4)

When do centrioles duplicate?

What is a licensing factor?
Speculate how Rogers et al. blocked Drosophila cells production of different proteins.
Make a pathway diagram that shows how the Science News article puts the protein players in line to
regulate centriole duplication.
5) What are TWO things that can happen to a protein AFTER production (post-translational modifications)
that are suggested to play a role in the story of centriole duplication?
Beyond Guiding questions:
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Biology 355

Project 5 Centrioles

Spring 2015

So far, the story suggests that centriole duplication relies on regulation of protein degradation. What are
tools you could use to determine if ubiquitin-mediated degradation is the mechanism for regulating your
favorite proteins stability?

What does regulation through post-translational modification offer that regulation through gene
expression does not?

B) INTRODUCE, This project starts in section so your TA will give you a brief overview of where we are going.
The lecture on Monday will continue to deepen this basic foundation.
C) BUILD, We have an image-guided activity to help you understand why we might care about regulating the
duplication of centrioles. Our images are available at the end of this project description. You might want to
convert these into note-taking files for class today.
D) SUMMARIZE, Your TA will wrap things up and instruct you about how you will be moving forward.

Part 2
A)

PREPARATION (Do in advance)

1 Find two papers (same research, different ways of describing the work).
a) One is found at PMID 22623721. This is a peer-reviewed primary research paper.
b) The other is found at http://www.tandfonline.com/doi/full/10.4161/worm.22497 . This is an open-access,
online journal. Obtain both papers.
c) Read BOTH abstracts and introduction associated with PMID 22623721.
2 Start a glossary of terms and acronyms. This will be critical. There is a LOT of data to consider in this
project! Acronyms are tricky to research online. For instance, the first hit you get when you type in SCF is State
College of Florida. So type in the acronym AND centriole (or AND cell biology) to narrow down you search.
B)

LECTURE (as usual, lecture slides will be posted in advance to facilitate note-taking).

C)

DISCUSS
1- Use information you gathered from discussion section as well as information from the PMID 22623721
papers discussion.
2- Discuss why we might care about this topic, what benefit will come from the research.
3- With your group, propose THREE agencies you might go to if you were researching this topic and you
wanted to obtain funding for your experiments. Write these three agencies on your midterm 2 index card.

Part 3
A)

PREPARATION (Do in advance)

1 Obtain the supplements to PMID 22623721s paper.

2 Compare all three pieces of this story, the main paper, the supplements to that paper and the open-access
version of the paper. Make a table that indicates the pieces of information that are in each source.

Biology 355

Project 5 Centrioles

Spring 2015

3 Examine the figure to the right. Determine what shapes could be

labeled by ZYG-1, LIN-23, SEL-10, SKR1/2, CUL-1 (proteins found
in C. elegans). Use the abstract and introduction of the paper to help
you. Diagram source http://www.celldiv.com/content/1/1/30
4 Come to class with a question you can ask Dr. Peel (first author on
the paper we are reading). In todays visit from the first author of our
paper (Dr. Peel from the College of New Jersy) well think a bit bigger
picture than previous visits (both Theo and Dr. Mosca came to class at
the conclusion of a project, Dr. Peel is coming prior to you reading the
entire paper). Consider the bigger picture topics of why we might care
about this work and who might fund it. Also consider topics pertaining
to the publication process. How do you pick a journal? How do you
decide what information goes into a paper, what gets saved, what goes
in supplements?
B) AUTHORS CORNER (20 minutes)
Q&A with Dr. Peel.
C) CHECK IN
D) TIPS (for managing some of the details in the paper)
E) DISCUSS
1 Use the title of the article (PMID 22623721), the abstract, and the headers for the results section.
2 Outline what information is being presented in this article that is new research findings.
3 Compare this outline to the figures is there data presented that is not on your outline?
4 Continue to build your vocabulary lists.
F) OBTAIN YOUR FIGURE ASSIGNMENT

Part 4
A)

PREPARATION (Do in advance)

1 Read the paper corresponding to PMID, focusing on the portions necessary to understand your figure.
2 BQMOC your figure (one sentence each portion). Bring that as evidence for your TA of your preparation.
3 In addition to BQMOCing your figure, you will build your OWN guiding questions. Guiding questions have
been present for you on previous projects. Doc Linda used them as bite-sized mini-questions to help you figure
out what was going on in the figure (or abstract or video). We offer you (below) the example figure 1. We have
BQMOCed it and offered figure 1 guiding questions. You should each write one guiding question, one per
individual in your group. You will be submitting these or revised ones to your colleagues who are studying a
different figure AND to your instructor (these, too, will be worth midterm exam points). For your peers, consider
using a Google Doc or Canvas to share your guiding questions. For Doc Linda you will be submitting the same
questions to CANVAS.
B) DISCUSS (15 minutes) Share your understanding of your figure with your group.
C) PRESENT (25 minutes) Presentation of each figure.
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Biology 355

Project 5 Centrioles

Spring 2015

D) WRAP UP (5 minutes)
Figure 1 example:
B: ZYG-1 levels are variable during the cell cycle.ZYG-1 is a kinase that is similar to SAK/PLK4 of Drosophila
which is important for regulating centriole duplication. SAK/PLK4 levels fluctuate during the cell cycle.
Q: Can ZYG-1 level fluctuation be demonstrated in single celled embryos? Does ZYG-1 antibody staining change
during the cell cycle? Does GFP::ZYG-1 fluorescence change during the cell cycle?
M: Immunofluorescence is quantified during the first cell cycle. ZYG-1 antibodies for fluctuations in endogenous
ZYG-1 and GFP fluorescence is used to quantify GFP::ZYG-1 levels.
O: ZYG-1 protein levels change during the first cell cycle, being lowest at prophase and highest at anaphase.
ZYG-1 antibody staining shows ~3 fold increase between prophase and anaphase. GFP::ZYG-1 levels increase ~
50% between prophase and anaphase.
C: There is significantly more ZYG-1 in anaphase cells as compared to prophase cells in the first cell cycle by
either method. The endogenous ZYG-1 levels show significant increases from prophase to metaphase and
significant increases between metaphase and anaphase. The GFP::ZYG-1 levels rise to a lesser degree (compared
to the endogenous ZYG-1) but show significant increases between prophase and anaphase.
Guiding questions:
1. What is producing the fluorescence in panel A as opposed to panel B? A fluorescent antibody to ZYG1, B GFP fused to ZYG-1 protein.
2. What does A.U. stand for, and how do you interpret this parameter? Arbitrary units. These are
measurements of fluorescence that can be compared WITHIN an experiment, one condition to the others.
They tell us about relative differences.
3. When, during development, are they examining ZYG-1 changes? Why is this important? During the first
cell division (from a single-celled embryo preparing to become a 2-cell embryo). This is only important
in that they can SYNCHRONIZE the cells and capture them all at the same relative time post fertilization
and know based on time WHERE in the cell cycle they are.
4. How does the GFP::ZYG-1 measure up against the endogenous ZYG-1 protein? Both show the same
relative upward trend, more protein in anaphase than prophase. The GFP fusion shows less overall
increase.

Part 5
A)

PREPARATION (Do in advance)

1 Complete your reading of PMID 22623721.

2 Go back to the supplements and the open-source pieces. Write a 1- paragraph summary of your OPINION on
what goes where and why. Submit this on paper to Doc Linda at the start of class. This (along with your guiding
questions for your figure) will count toward your second midterm exam. If you cannot come to class, please
submit your paragraph by email by the start of class to have it count for full credit.
3 Obtain the guiding questions from your discussion section peers. If you used a GOOGLE DOC or CANVAS
there is no need for printing. Use the guiding questions to help you finish reading the paper and analyze the
figures.
4 Study figures 6 and 7.
B) INTRODUCTION: Where are we, what do we know, what do we need to know?
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Biology 355

Project 5 Centrioles

Spring 2015

C) DISCUSSION:
1 Answer the guiding questions from your peers to review and deepen your understanding of the results shown
in the paper.
2 We did not analyze figures 6 and 7 yet. Start with figure 7, as that is fairly straight-forward if you got CoIP
from project 3. Go over your understanding with your team as this will be a question on the next midterm exam.
Then go over figure 6 and ensure you know what the research QUESTION is for this figure. Put this on your
index card for midterm 2.
D) WRAP UP
E) AFTERMATH
End-of-project study guide.
End-of-project quiz.
Executive summary (on figure 2, 3, 4, or 5 but NOT the figure you presented in class). OPTIONAL you may
only submit 3 executive summaries total, we do not allow you to submit all four and drop your lowest grade.
Critical thinking questions:
1) In 2009, two papers were published nearly simultaneously on the same regulatory pathway for Drosophila
centrioles. Find the PMIDs for these papers and put them on your midterm 2 index card.
2) For both of these papers, the ZYG-1 equivalent is named PLK4 or SAK (same protein, different names).
This protein has a mutational variant that cannot bind to the F-box protein of the SCF. This mutation is
called the SBM (slimb-binding mutant) or SAK-ND (SAK non-degradable). What changes in this
mutation?
3) SAK/PLK4 is a kinase. What does the SBM (or SAK-ND) mutation suggest about another posttranslational modification that happens to SAK to regulate its activity?
4) What protein is family is found in the cytoskeleton AND the centriole? If you wanted to inhibit this
protein what would you use? What future question could you ask by testing cells with this inhibitor?
5) There is a lot of genetics here. Compare these specific alleles: which is unlike the other two? zyg-1
(it25), lin-23 (otl) and sel-10(ok1632).
6) Why might there be TWO F-box proteins regulating ZYG-1? How do the authors test if the proteins are
redundant?
7) What are cyclins? What is cyclin E? How is cyclin E important in this study?
8) Name / describe TWO ways that a cell can regulate the activity of a protein (post translationally, not in
production). How does each of these ways work to enable centrioles to function and divide properly?
IMAGES for Part 1 discussion, enlarge as you find useful for note-taking in section

Biology 355

Project 5 Centrioles

Spring 2015

Biology 355

Project 5 Centrioles

you want integer numbers for ploidy to ensure fitness.

Spring 2015

Biology 355

Project 5 Centrioles

Spring 2015

Biology 355

Project 5 Centrioles

Spring 2015

9 by 3 array microtubules of in centrioles.

Gamma tubulin helps nucleate centriole. Starting material, a and beta grows.
Pcm, bunch of really good proteins
Microtubule spindle assembly.
G1, s phase, mitosis. Centriole doublet separates in g1, two new doublets formed. Starts in s
phase, finishes in m phase. M phase allows for spindle fibers to appear.
Post translational modification, phosphorylation, glycosylation, ubiquityaltion, sumoylation.
Cant assume phosphate means on position
It means off.