Activation of the apoptotic pathway during prolonged prometaphase blocks daughter cell proliferation.

When untransformed human cells spend >1.5 h in prometaphase under standard culture conditions, all daughters arrest in G1 despite normal division of their mothers. We investigate what happens during prolonged prometaphase that leads to daughter cell arrest in the absence of DNA damage.

Sophisticated lessons from simple organisms: appreciating the value of curiosity-driven research.

For hundreds of years, biologists have studied accessible organisms such as garden peas, sea urchins collected at low tide, newt eggs, and flies circling rotten fruit. These organisms help us to understand the world around us, attracting and inspiring each new generation of biologists with the promise of mystery and discovery. Time and time again, what we learn from such simple organisms has emphasized our common biological origins by proving to be applicable to more complex organisms, including humans.

The Zn-finger domain of MdmX suppresses cancer progression by promoting genome stability in p53-mutant cells.

The MDMX (MDM4) oncogene is amplified or overexpressed in a significant percentage of human tumors. MDMX is thought to function as an oncoprotein by binding p53 tumor suppressor protein to inhibit p53-mediated transcription, and by complexing with MDM2 oncoprotein to promote MDM2-mediated degradation of p53. However, down-regulation or loss of functional MDMX has also been observed in a variety of human tumors that are mutated for p53, often correlating with more aggressive cancers and a worse patient prognosis.

Live Cell Imaging: Assessing the Phototoxicity of 488 and 546 nm Light and Methods to Alleviate it.

In live cell imaging of fluorescent proteins, phototoxicity of the excitation light can be problematical. Cell death is obvious, but reduced cell viability can make the interpretation of observations error prone. We characterized the phototoxic consequences of 488 and 546 nm light on untransformed human cells and tested methods that have or could be used to alleviate photodamage.

Using sea urchin gametes and zygotes to investigate centrosome duplication.

Centriole structure and function in the sea urchin zygote parallel those in mammalian somatic cells. Here, I briefly introduce the properties and attributes of the sea urchin system that make it an attractive platform for the study of centrosome and centriole duplication. These attributes apply to all echinoderms readily available from commercial suppliers: sea urchins, sand dollars, and starfish.

A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis.

Precise regulation of centrosome number is critical for accurate chromosome segregation and the maintenance of genomic integrity. In nontransformed cells, centrosome loss triggers a p53-dependent surveillance pathway that protects against genome instability by blocking cell growth. However, the mechanism by which p53 is activated in response to centrosome loss remains unknown.

p53 protects against genome instability following centriole duplication failure.

Centriole function has been difficult to study because of a lack of specific tools that allow persistent and reversible centriole depletion. Here we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centriole biogenesis. Depletion of Plk4 led to a failure of centriole duplication that produced an irreversible cell cycle arrest within a few divisions.

The SWI/SNF ATPases Are Required for Triple Negative Breast Cancer Cell Proliferation.

The Brahma (BRM) and Brahma-related Gene 1 (BRG1) ATPases are highly conserved homologs that catalyze the chromatin remodeling functions of the multi-subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in many cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status.

One to only two: a short history of the centrosome and its duplication.

This review discusses some of the history of the fundamental, but not fully solved problem of how the centrosome duplicates from one to only two as the cell prepares for mitosis. We start with some of the early descriptions of the centrosome and the remarkably prescient but then controversial inferences drawn concerning its function in the cell. For more than 100 years, one of the most difficult issues for the concept of the centrosome has been to integrate observations that centrosomes appear to be important for spindle assembly in animal cells yet are not evident in higher plant cells and some animal cells.

Link between DNA damage and centriole disengagement/reduplication in untransformed human cells.

The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells.

Practical aspects of adjusting digital cameras.

This chapter introduces the adjustment of digital camera settings using the tools found within image acquisition software and discusses measuring gray-level information such as (1) the histogram, (2) line scan, and (3) other strategies. The pixel values in an image can be measured within many image capture software programs in two ways. The first is a histogram of pixel gray values and the second is a line-scan plot across a selectable axis of the image.

Microscope basics.

This chapter provides information on how microscopes work and discusses some of the microscope issues to be considered in using a video camera on the microscope. There are two types of microscopes in use today for research in cell biology-the older finite tube-length (typically 160mm mechanical tube length) microscopes and the infinity optics microscopes that are now produced. The objective lens forms a magnified, real image of the specimen at a specific distance from the objective known as the intermediate image plane.

Centriole engagement: it's not just cohesin any more.

The belief that cohesin complexes link mother to daughter centrioles has received substantial experimental support. New studies challenge the primacy of cohesin in centriole engagement and provide a more nuanced view into the mechanisms for centriole disengagement in anaphase.

The interrelationship between APC/C and Plk1 activities in centriole disengagement.

Mother-daughter centriole disengagement, the necessary first step in centriole duplication, involves Plk1 activity in early mitosis and separase activity after APC/C activity mediates securin degradation. Plk1 activity is thought to be essential and sufficient for centriole disengagement with separase activity playing a supporting but non-essential role. In separase null cells, however, centriole disengagement is substantially delayed.

Repeated cleavage failure does not establish centrosome amplification in untransformed human cells.

We tested whether cleavage failure as a transient event establishes an incidence of centrosome amplification in cell populations. Five rounds of ∼30% cytochalasin-induced cleavage failure in untransformed human cell cultures did not establish centrosome amplification in the short or long terms. The progeny of binucleate cells progressively dropped out of the cell cycle and expressed p53/p21, and none divided a fourth time.

Centriole duplication: analogue control in a digital age.

In preparation for mitosis, the centrosome doubles once and only once to provide the two poles of the mitotic spindle. The presence of more than two centrosomes increases the chances that mitosis will be multipolar, and chromosomes will be distributed unequally. Since the number of mother-daughter centriole pairs determines the number of centrosomes, it is important that only one daughter centriole is assembled at, but slightly separated from, the proximal end of each mother centriole.

Prolonged prometaphase blocks daughter cell proliferation despite normal completion of mitosis.

The mitotic checkpoint maintains genomic stability by blocking the metaphase-anaphase transition until all kinetochores attach to spindle microtubules [1, 2]. However, some defects are not detected by this checkpoint. With low concentrations of microtubule-targeting agents, the checkpoint eventually becomes satisfied, though the spindles may be short and/or multipolar [3, 4] and the fidelity of chromosome distribution and cleavage completion are compromised.

Abrogation of the postmitotic checkpoint contributes to polyploidization in human papillomavirus E7-expressing cells.

High-risk types of human papillomavirus (HPV) are considered the major causative agents of cervical carcinoma. The transforming ability of HPV resides in the E6 and E7 oncogenes, yet the pathway to transformation is not well understood. Cells expressing the oncogene E7 from high-risk HPVs have a high incidence of polyploidy, which has been shown to occur as an early event in cervical carcinogenesis and predisposes the cells to aneuploidy.

MdmX regulates transformation and chromosomal stability in p53-deficient cells.

The cellular homologues Mdm2 and MdmX play critical roles in regulating the activity of the p53 tumor suppressor in damaged and non-damaged cells and during development in mice. Recently, we have utilized genetically defined primary cells and mice to reveal that endogenous levels of MdmX can also suppress multipolar mitosis and transformation in hyperploid p53-deficient cells and tumorigenesis in p53-deficient mice. These MdmX functions are not shared by Mdm2, and are distinct from the well-established ability of MdmX to complex with and inhibit p53 activity.

Cyclin E in centrosome duplication and reduplication in sea urchin zygotes.

When protein synthesis is completely blocked from before fertilization, the sea urchin zygote arrests in first S phase and the paternal centrosome reduplicates multiple times. However, when protein synthesis is blocked starting in prophase of first mitosis, the zygote divides and the blastomeres arrest in a G1-like state. The centrosome inherited from this mitosis duplicates only once in each blastomere for reasons that are not understood.

Cell-cycle progression without an intact microtuble cytoskeleton.

For mammalian somatic cells, the importance of microtubule cytoskeleton integrity during interphase cell-cycle progression is uncertain. The loss, suppression, or stabilization of the microtubule cytoskeleton has been widely reported to cause a G1 arrest in a variable, and often high, proportion of cell populations, suggesting the existence of a "microtubule damage," "microtubule integrity," or "postmitotic" checkpoint in G1 or G2. We found that when normal human cells (hTERT RPE1 and primary fibroblasts) are continuously exposed to nocodazole, they remain in mitosis for 10-48 hr before they slip out of mitosis and arrest in G1; this finding is consistent with previous reports.

Centriole biogenesis: a tale of two pathways.

Two recent studies in demonstrate that overexpression of proteins required for centriole duplication can not only induce centriole over-duplication in cells containing centrioles, but can also drive centriole assembly in unfertilized eggs that initially lack centrioles. These studies offer a new perspective on the mechanisms that control centriole duplication.