Centrosomes nucleate most spindle microtubules and thus, determine spindle polarity. Since the essential bipolarity of mitosis depends upon the cell containing just two centrosomes, centrosome duplication must be under tight numerical and temporal control. Cleavage failure and centrosome reduplication are thought to be the most probable causes of centrosome amplification (>2 centrosomes at mitosis). Extra centrosomes raise he chances for spindle multipolarity and consequent unequal chromosome distribution. This leads to the aneuploidy and genomic instability that drives the evolution of the transformed state. Our research uses individual living cells to investigate the controls for cell division with an emphasis on the interrelationship between centrosomes and the cell cycle.
Aim 1 : We will characterize the process of centriole/centrosome reduplication during prolonged S phase in living CHO and U2OS cells. We also will characterize human papillomavirus oncoprotein E7 induction of centrosome amplification.
Aim 2 : We will investigate the importance of centrosome localization sequence (CLS) dependent binding of cyclins A and E to the centrosome for the duplication and reduplication of the centrosome.
Aim 3 : For normal human cells we will characterize the functional consequences of cleavage failure in the genesis of persistent centrosome amplification and investigate the proliferative capacity of tetraploid cells. We will determine if cleavage failure can produce persistent centrosome amplification in CHO cells that have a mutated p53 and do not arrest when tetraploid.
Aim 4 : We will investigate the relationship between the duration of mitosis and the ability of daughter cells to progress through G1 in normal human cells. We will investigate why a modest (>1 hour) prolongation of mitosis causes the daughter cells of a seemingly normal division to arrest in G1.
Aim 5 : We will investigate whether centriole structure is determined by the self-assembly characteristics of its subunits or by a template present at the mother centriole. We will introduce C. elegans (worm) centrioles with 9 singlet microtubules into Xenopus (frog) egg extracts that support duplication of centrioles with 9 triplet microtubules. We will determine if worm centrioles are functional in a heterologous cytoplasm and examine the ultrastructure of the daughter centrioles to determine if they have the worm, the frog, or some other structure. ? ? ?
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