Cellular Dynamics
Section Leader
Edward H. Hinchcliffe, Ph.D.
Associate Professor

The Hormel Institute - Edward Hinchcliffe

Our research section is funded by grants from the National Institutes of Health, and the Department of Defense CDMRP (Congressionally Directed Medical Research Programs). We study the regulation of cell division . the process by which cells proliferate. We have several ongoing research projects in the lab, including understanding the molecular mechanisms underlying the generation of mitotic spindle bipolarity, and the gain/loss of whole chromosomes during mitotic division, a process which is associated with tumor progression.

 

Cell division lies at the heart of normal tissue development and maintenance. The division of cells must occur in a strict one-to-two fashion to ensure genomic stability. The loss or gain of whole chromosomes during abnormal cell division leads to aneuploidy in which daughter cells have variable chromosome numbers. This is a major problem for cells because there is a change in the dosage of essential gene products. The cell has developed multiple biochemical checkpoints and failsafe devices to ensure that cell division occurs with absolute fidelity. Unfortunately, DNA mutations . often caused by environmental factors can render these molecular quality control mechanisms inoperable. The result is the inadvertent missegregation of chromosomes during cell division, leading to genomic abnormalities and tumorigenesis.

Chromosome instability (CIN) is a hallmark of solid tumors and contributes to the genomic heterogeneity of tumor cells. There are multiple mechanisms believed to underlie the generation of CIN, including cell cycle defects, abnormal centrosome duplication and function, premature chromatid disjunction, and centrosome separation errors. However, despite an increasingly mechanistic understanding of how CIN is generated, we know relatively little about how chromosome missegregation becomes transduced into cell transformation and tumorigenesis. A major unresolved question is the role of cell cycle checkpoints and failsafe devices in preventing chromosome missegregation in the first place. The question of how a single missegregated chromosome can trigger the p53/ p21 pathway and induce durable cell cycle arrest . a molecular failsafe device that monitors aneuploidy and prevents the proliferation of aneuploid cells. Current work focused on DNA damage caused by lagging chromosomes is part of the answer. To date, no mechanisms, however, have been identified that monitor chromosome mispositioning . either before or after anaphase . at the singlechromosome level.

The centrosome is an organelle that nucleates and organizes the microtubule cytoskeleton. This, in turn, is used to build the bipolar mitotic spindle, which is responsible for aligning and segregating the duplicated chromosomes during cell division. Centrosomes are thought to play a major role in establishing the bipolarity of the mitotic spindle. To ensure this, the single centrosome normally duplicates exactly once during the cell cycle, yielding a pair of centrosomes that form the two spindle poles. In many cancer cells, the number of centrosomes increases, resulting in a small but significant number of cells with more than two spindle poles and an increase in the probability of abnormal cell division. Therefore, it is important to understand the molecular mechanisms that drive normal centrosome duplication, and importantly, restrict centrosome duplication to once per cell cycle.

In our lab, we use cultured mammalian cells and cytoplasmic extracts generated from Xenopus frogs to examine the basic control mechanisms underlying centrosome duplication, cell division, and cytokinesis. We use advanced imaging techniques, such as live-cell confocal fluorescence microscopy, Fluorescence Recovery After Photobleaching (FRAP), microinjection, and microsurgery to address these fundamental questions in cell biology. Our research has direct relevance to understanding the underlying mechanisms that lead to cancer initiation and progression. Our work also is relevant to identifying potential targets for chemotherapy agents

“Mistakes in the cell-division process can have disastrous
consequences for the cell, leading to aneuploidy, cellular
transformation and tumorogenesis.”
Dr. Edward H. Hinchcliffe

Experimental research results

1. Chromosome missegregation: Contributing to the onset of tumorigenesis
Our long-term goal is to understand the cell cycle regulation of bipolar mitotic spindle assembly and function. Proper bipolar mitotic spindle assembly ensures that each daughter cell receives an exact set of chromosomes. Chromosome instability (CIN) . the loss or gain of individual chromosomes during mitosis . generates aneuploidy, and correlates with the aggressive behavior of advanced tumor cells. Recent studies have linked chromosome segregation errors to merotelic kinetochore attachments caused by transient defects in spindle geometry, often mediated by supernumerary centrosomes. Yet despite our increasingly mechanistic understanding of the causes of CIN, the important question of how both transformed and non-transformed cells respond to chromosome instability remains poorly understood. To this end, we recently have identified a novel biochemical pathway that monitors chromosome missegregation. We find that misaligned chromosomes (i.e. those well away from the metaphase plate) activate a dynamic positional .sensor., involving phosphorylation of the highly conserved histone variant H3.3. H3.3 differs from the canonical H3.1 by 5 AA substitutions; one of which, Ser 31 is phosphorylated only during mitosis (Ser31-P). Whereas all congressed chromosomes have Ser31-P confined to their peri-centromeric regions, we find that misaligned chromosomes accumulate Ser31-P along their arms. H3.3 Ser31 hyper-phosphorylation persists after anaphase, and is found on both lagging chromosomes in the bridge, and disjoined pairs of chromatids synteliclyattached to one pole. Thus, Ser31-P serves as a dynamic mark for CIN in both mitotic and post-mitotic cells. We are characterizing the Ser31 phosphorylation pathway used to recognize misaligned chromosomes. We are determining the mechanism used to generate the Ser31-P proximity sensor on misaligned chromosomes, and identify both the kinase and the phosphatase responsible for generating this dynamic mark. We are using live-cell imaging assays to determine the fate of cells that exit mitosis with missegregated chromosomes while simultaneously using biochemical/genetic methods to inactivate Ser31 phosphorylation in these cells. We are testing whether H3.3 Ser31P affects cell fate or proliferation in CIN cells. Recent work has shown that single nucleotide somatic mutations in the tail of the H3.3 gene (K27M and G34R) are associated with human cancers. Both mutations flank Ser31. We will test the role of these flanking AA substitutions in modulating H3.3 Ser31-P, and in the ability of H3.3 to bind potential regulatory elements. Our work is innovative because is capitalizes on a novel pathway to identify chromosome missegregation in individual cells. It is also important because, for the first time, it allows for the biochemical manipulation of basic cellular responses to chromosome missegregation and aneuploidy.

The Hormel Institute - Edward Hinchcliffe Lab

(Left to right) Charles (Charlie) Day, Edward Hinchcliffe, Alyssa Langfald, Sela Fadness
Not pictured: Kul Karanjeet

2. Building a bipolar spindle
Mitosis must be carried out with high fidelity to ensure that each daughter cell receives a complete compliment of the genome. Mistakes in the cell division process can have disastrous consequences for the cell . leading to aneuploidy, cellular transformation and tumorogenesis. The centrosome is known to play a critical structural role in the cell division process . it organizes the microtubule network during interphase and astral microtubules at the spindle poles during mitosis.

We currently are using microsurgery coupled with time-lapse videomicroscopy of living acentrosomal cells to investigate the role of the centrosome in cell cycle regulation. To directly visualize the role of microtubules, and regulatory molecules during the acentrosomal cell cycle, we have generated primate kidney cell line (BSC-1 cells) that constitutively express .-tubulin coupled to GFP. We find that after several hours, acentrosomal cells re-form their microtubule network into an organized array. Interestingly, the acentrosomal microtubule focus can separate into two distinct poles prior to nuclear envelope breakdown. This demonstrates that the splitting of the microtubule network does not require a centrosome, contrary to previously held notions. However, we find that in the absence of a centrosome, the splitting of the microtubule network is inefficient; ~40% of acentrosomal cells enter mitosis with a monopolar spindle. These cells cannot bipolarize and fail cytokinesis. Thus, there is some aspect of the centrosome that ensures that the microtubule network will split and separate before the onset of mitosis. It could be that acentrosomal microtubule focus is deficient in the recruitment of some key factor(s) necessary to ensure accurate splitting. This factor could be a regulatory activity, a structural activity, or a combination of the two. It is also possible that the acentrosomal microtubule focus lacks sufficient microtubules to interact with the cell cortex. Regardless of the mechanism, our work reveals that centrosomes are absolutely necessary to ensure fidelity during mitotic spindle assembly.

3. Tektin proteins: key to spindle poles and spindle midbodies
We currently are investigating the role of the tektin proteins in establishing the spindle midzone. Tektins were first identified as components of axonemal microtubules, where they are thought to impart structural rigidity and complex periodic spacing to these highly stable microtubules. Our recent results suggest that tektins localize to the overlapping microtubules at the spindle midzone, where they also play an important role in the spindle midzone. This region of the mitotic spindle is responsible for initiating cytokinesis and required for the process to continue. Many key regulators of late mitotic events, along with cytokinesis localize to the spindle midzone. When tektin 2 (one of three distinct tektins found in vertebrates) is knocked-down using shRNAs, the midzone microtubules fail to become compacted, and appear to exhibit abnormal plusend microtubule motility. The result is failure of cytokinesis.

In addition to the tektins, we are exploring the role of two highly conserved proteins called EFHC 1 & 2. These Ca2+-binding proteins also are involved in centriole assembly and the formation of midbodies. Importantly, mutations in these proteins lead to abnormal cell division, associated with neurological birth defects.

We are interested in uncovering the molecular mechanisms underlying these observations. We currently are examining the motility of several key regulators of midzone function: PRC1 and Kif4, in response to experimental loss of tektin 2 and or EFHC 1&2. We are using live-cell imaging, and FRAP analysis to examine the role played by tektins in regulating these important components of the cell division apparatus.

4. Coordinating cytokinetic furrow formation with anaphase onset
The cell-division furrow . created by the recruitment of actin filaments and the motor protein myosin II . is formed between the separating sister chromatids at anaphase. This furrow constricts the dividing cell into two daughters. To ensure that cytokinesis occurs in the right place and at the right time, the positioning of the cleavage furrow must be coupled to the segregation of the chromosomes. This occurs through signaling via the microtubule network, specifically the dynamic astral microtubules and the stable overlapping midzone microtubules. Both of these classes of microtubules are important for signaling the formation of the cytokinetic furrow and for ensuring that the furrow remains restricted to the cell center. We are investigating the regulation of furrow formation using live-cell imaging and single-cell manipulation. We are taking advantage of the fact that microtubules are extremely sensitive to temperature and can be disassembled by cold treatment without causing harm to the cell. When the cells are warmed up, the microtubule re-assemble and the cell cycle proceeds on its way. Using this system and spinning disk confocal microscopy, we are able to examine the roles of candidate regulatory mechanisms, including Aurora B kinase, Polo-like kinase 1, and the relative contributions of the astral and midzone microtubules. Our goal is to integrate molecular studies with livecell physiology to understand the mechanisms underlying cell division.

We have found that there is a period following the onset of anaphase where the cell cortex can respond to furrow-inducing signals, and this period is sensitive to the loss of microtubules and the activity of Polo-like kinase 1. Once cells progress beyond this point, however, the furrow will form, regardless of whether or not microtubules persist. Polo-like kinase 1 activity also is not required after this .point of no return;. adding kinase inhibitors after this point does not affect the ability of a furrow to assemble.

A detailed understanding of the regulation of cell division, cytokinesis, and chromosome instability will advance our knowledge of the biology of cancer . itself a disease characterized by unregulated cell proliferation and chromosome missegregation. Our work will provide for a mechanistic understanding of key cell cycle events that may contribute to cancer progression. Together, these studies also will provide a source of potential targets for future anti-cancer drugs.

Funding
Department of Defense (CDMRP), CA130436
National Institutes of Health, R01HL125353

Other professional activities
Review panel, National Science Foundation, USA Mentor, American Society for Cell Biology Minorities Affairs Committee, FRED mentor

Ad hoc reviewer:
European Research Council Medical Research Council, UK Wellcome Trust, UK