Miller Lab Research
How does a non-sentient protein assembly make decisions?
Have you ever wondered how molecules, proteins, and cells without brains accomplish their very detailed tasks? How do they know when to do what?
The kinetochore is the protein machine responsible for ensuring accurate chromosome segregation during cell division. It assimilates signals and cues from chemical messengers, cell cycle stage, its own position within the cell, and how much tension it is under. With all that information, it determines when the cell is ready to progress through division. It not only decides whether the cell is ready or not to continue dividing, but also physically links the chromosomes to the microtubules that will ultimately provide the driving force for pulling sister chromatids apart.
Microtubule tips are also an important facet of this entire process, but are so dynamic that we still know relatively little about the interactions and processes occurring at the microtubule tip, where a kinetochore may be bound.
We aim to understand how such large, dynamic, and complicated protein machines can direct the most fundamental process of life: passing on genetic information, with such accuracy that a person’s body can undergo billions of cell divisions throughout their life, while only rarely making mistakes.
Current Research
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Kinetochores “sense” whether correct kinetochore-microtubule attachments have been made based on tension. Erroneous low-tension attachments are sensed through an unknown mechanism (or mechanisms), and are then selectively destabilized and released, allowing the cell to try again. We identified the first critical factor in a mechanical tension sensing pathway, Stu2, and are working to understand how it performs this crucial task.
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Although the kinetochore-microtubule interface is crucial, the microtubule side is surprisingly understudied. Even cutting-edge in vitro structures are unable to capture the range of interactions needed for this dynamic process in cells. Yeast genetics provide a powerful workhorse to uncover these interactions and to understand how mutations may impact chromosome segregation in the highly conserved tubulin proteins.
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Errors in kinetochore-microtubule attachments, among other mitotic errors, can cause chromosomes to remain trapped between dividing cells. As the cells move apart, the trapped chromosomes experience dangerously high stretching forces which, if left unresolved, can cause the chromosomes to break or even shatter. We are currently investigating a mechanism involving ESCRT-III family proteins by which cells may protect DNA from breakage due to high tension.
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We developed high-throughput CRISPR-based tiling mutagenesis platforms to systematically perturb protein complexes and identify the residues, interfaces, and mechanisms that underlie their function. By "breaking" molecular machines in thousands of precise ways, we uncover the design principles that govern their activity.
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Through complex signaling, cells control their progression through the cell cycle in response to cues indicating if the cell is prepared for the next phase. We are studying a central regulator of this process, Mps1, which performs a multitude of tasks to orchestrate this process, including destabilizing incorrect kinetochore attachments.
Our Approach…es
Our lab takes an interdisciplinary approach to our studies, using a combination of tools and techniques taken from many different disciplines. Yeast, our model organism, has a plethora of tools available due to its long use as a model organism, and we take full advantage of all these tools to answer scientific questions.
For example:
We use in vitro reconstitution assays to test biophysical aspects of kinetochores on our optical trap
We combine yeast genetics with cell biology to watch cellular functions in live microscopy
We have further developed CRISPR screening methods to quickly and thoroughly identify the most interesting mutations in the kinetochore
We mutate kinases involved in chromosome segregation and then study the effects on phosphorylation of the kinetochore
Watch a silly animated story about our favorite protein machine
Watch Matt explain his research journey and our lab goals
