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. We find that progressive loss of anti-apoptotic MCL-1 activity and oxidative stress act in concert to partially activate the apoptosis pathway, resulting in the delayed death of some daughters and senescence for the rest. At physiological oxygen levels, longer prometaphase durations are needed for all daughters to arrest. Partial activation of apoptosis during prolonged prometaphase leads to persistent caspase activity, which activates the kinase cascade mediating the post-mitotic activation of p38. This in turn activates p53, and the consequent expression of p21stops the cell cycle. This mechanism can prevent cells suffering intractable mitotic defects, which modestly prolong mitosis but allow its completion without DNA damage, from producing future cell generations that are susceptible to the evolution of a transformed phenotype.

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. Yet, biologists are increasingly being tasked with developing applications from the known, rather than being allowed to follow a path to discovery of the as yet unknown. Here, we provide examples of important lessons learned from research using selected non-vertebrate organisms. We argue that, for the purpose of understanding human disease, simple organisms cannot and should not be replaced solely by human cell-based culture systems. Rather, these organisms serve as powerful discovery tools for new knowledge that could subsequently be tested for conservation in human cell-based culture systems. In this way, curiosity-driven biological research in simple organisms has and will continue to pay huge dividends in both the short and long run for improving the human condition.

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. Unlabeled RPE1 cells were given single 0.5-2.5 min irradiations in early G1 from a mercury arc lamp on a fluorescence microscope. Four hundred eighty-eight nanometer light produced a dose-dependent decrease in the percentage of cells that progressed to mitosis, slowing of the cell cycle for some of those entering mitosis, and a ∼12% incidence of cell death for the highest dose. For 546 nm light we found a 10-15% reduction in the percentage of cells entering mitosis, no strong dose dependency, and a ∼2% incidence of cell death for the longest irradiations. For cells expressing GFP-centrin1 or mCherry-centrin1, fewer entered mitosis for each dose than unlabeled cells. For constant total dose 488 nm light irradiations of unlabeled cells, reducing the intensity 10-fold or spreading the exposures out as a series of 10 sec pulses at 1 min intervals produced a minor and not consistent improvement in the percentage of cells entering mitosis. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Trolox noticeably increased the fraction of cells entering mitosis. Thus, for long-term imaging there can be value to using RFP constructs and for GFP-tagged proteins reducing oxidative processes. J. Cell. Physiol. 232: 2461-2468, 2017. © 2016 Wiley Periodicals, Inc.

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. I list some of the practical aspects of the system that make it a cost- and time-effective system for experimental work and then list properties that are a "tool kit" that can be used to conduct studies that would not be practical, or in some cases not possible, with mammalian somatic cells. Since centrioles organize and localize the pericentriolar material that nucleates the astral arrays of microtubules (Bobinnec et al. in J Cell Biol 143(6):1575-1589, 1998), the pattern of aster duplication over several cell cycles can be used as a reliable measure for centriole duplication (Sluder and Rieder in J Cell Biol 100(3):887-896, 1985). Descriptions of the methods my laboratory has used to handle and image echinoderm zygotes are reviewed in Sluder et al. (Methods Cell Biol 61:439-472, 1999). Also included is a bibliography of papers that describe additional methods.

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. Here, we have used genome-wide CRISPR/Cas9 knockout screens to identify a USP28-53BP1-p53-p21 signaling axis at the core of the centrosome surveillance pathway. We show that USP28 and 53BP1 act to stabilize p53 after centrosome loss and demonstrate this function to be independent of their previously characterized role in the DNA damage response. Surprisingly, the USP28-53BP1-p53-p21 signaling pathway is also required to arrest cell growth after a prolonged prometaphase. We therefore propose that centrosome loss or a prolonged mitosis activate a common signaling pathway that acts to prevent the growth of cells that have an increased propensity for mitotic errors.

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. This arrest was not a result of a prolonged mitosis, chromosome segregation errors, or cytokinesis failure. Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely. Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate. In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure.

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. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence, or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in the loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells.

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. This stirred debate over the existence of centrosomes and their importance. A parallel debate concerned the role of the centrioles in organizing centrosomes. The relatively recent elucidation of bipolar spindle assembly around chromatin allows a re-examination of the role of centrioles in controlling centrosome duplication in animal cells. The problem of how centrosomes precisely double in preparation for mitosis in animal cells has now moved to the mystery of how only one procentriole is assembled at each mother centriole.

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. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 h, though 14% of the cells eventually go through mitosis in that time. By 72 h after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53-dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations.

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.

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. All objectives are designed to be used with the specimen at a defined distance from the front lens element of the objective (the working distance) so that the image formed is located at a specific location in the microscope. Infinity optics microscopes differ from the finite tube-length microscopes in that the objectives are designed to project the image of the specimen to infinity and do not, on their own, form a real image of the specimen. Three types of objectives are in common use today-plan achromats, plan apochromats, and plan fluorite lenses. The concept of mounting video cameras on the microscope is also presented in the chapter.

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. Understanding how to evaluate the information presented by these tools is critical to properly adjusting the camera to maximize the image contrast without losing grayscale information. This chapter discusses the 0-255 grayscale resolution of an 8-bit camera; however, the concepts are the same for cameras of any bit depth. This chapter also describes camera settings, such as exposure time, offset, and gain, and the steps for contrast stretching such as setting the exposure time, adjusting offset and gain, and camera versus image display controls.

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. The ability of APC/C activity alone to mediate centriole disengagement has not been directly tested. We investigate the interrelationship between Plk1 and APC/C activities in disengaging centrioles in S or G2 HeLa and RPE1 cells, cell types that do not reduplicate centrioles when arrested in S phase. Knockdown of the interphase APC/C inhibitor Emi1 leads to centriole disengagement and reduplication of the mother centrioles, though this is slow. Strong inhibition of Plk1 activity, if any, during S does not block centriole disengagement and mother centriole reduplication in Emi1 depleted cells. Centriole disengagement depends on APC/C-Cdh1 activity, not APC/C-Cdc20 activity. Also, Plk1 and APC/C-Cdh1 activities can independently promote centriole disengagement in G2 arrested cells. Thus, Plk1 and APC/C-Cdh1 activities are independent but slow pathways for centriole disengagement. By having two slow mechanisms for disengagement working together, the cell ensures that centrioles will not prematurely separate in late G2 or early mitosis, thereby risking multipolar spindle assembly, but rather disengage in a timely fashion only late in mitosis.

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. We also tested whether cleavage failure established centrosome amplification in transformed cell populations. Tetraploid HCT116 p53(-/-) cells eventually all failed cleavage repeatedly and ceased proliferating. HeLa cells all died or arrested within four cell cycles. Chinese hamster ovary cells proliferated after cleavage failure, but five rounds of induced cleavage failure produced a modest increase in the incidence of centrosome amplification in the short term, which did not rise with more cycles of cleavage failure. This incidence dropped to close to control values in the long term despite a 2-6% rate of spontaneous cleavage failure in the progeny of tetraploid cells.

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. This numerical and spatial specificity has led to the belief that a 'template' on the mother centriole provides a unique site for procentriole assembly. We review observations that are leading to the demise of this intuitively attractive idea. In its place, we are left with the notion that pericentriolar material at the wall of the mother centriole provides a local environment that promotes the assembly of a macromolecular complex that seeds the daughter centriole. Even though the system normally behaves in a digital fashion to go from zero to just one daughter centriole per mother, this behaviour appears to be based in the precise analogue control of multiple proteins, their activities, and the structure provided by the 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. In real life, environmental toxins, radiation, or chemotherapeutic agents may lead to completed but inaccurate mitoses. It has been assumed that once the checkpoint is satisfied and cells divide, the daughter cells would proliferate regardless of prometaphase duration. However, when continuously exposed to microtubule inhibitors, untransformed cells eventually slip out of mitosis after 12-48 hr and arrest in G1 [5-8] (see also [9]). Interestingly, transient but prolonged treatments with nocodazole allow completion of mitosis, but the daughter cells arrest in interphase [10, 11] (see also [9, 12]). Here we characterize the relationship between prometaphase duration and the proliferative capacity of daughter cells. Our results reveal the existence of a mechanism that senses prometaphase duration; if prometaphase lasts >1.5 hr, this mechanism triggers a durable p38- and p53-dependent G1 arrest of the daughter cells despite normal division of their mothers.

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. The mechanism through which E7 contributes to polyploidy is not known. It has been hypothesized that E7 induces polyploidy in response to mitotic stress by abrogating the mitotic spindle assembly checkpoint. It was also proposed that E7 may stimulate rereplication to induce polyploidy. We have tested these hypotheses by using human epithelial cells in which E7 expression induces a significant amount of polyploidy. We find that E7-expressing cells undergo normal mitoses with an intact spindle assembly checkpoint and that they are able to complete cytokinesis. Our results also exclude DNA rereplication as a major mechanism of polyploidization in E7-expressing cells upon microtubule disruption. Instead, we have shown that while normal cells arrest at the postmitotic checkpoint after adaptation to the spindle assembly checkpoint, E7-expressing cells replicate their DNA and propagate as polyploid cells. Thus, abrogation of the postmitotic checkpoint leads to polyploidy formation in E7-expressing human epithelial cells. Our results suggest that downregulation of pRb is important for E7 to induce polyploidy and abrogation of the postmitotic checkpoint.

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. Here we discuss some of the ramifications of MdmX loss in p53-deficient cells and mice, and we explore further the fate of MdmX/p53-double null embryonic fibroblasts undergoing multi-polar cell division using time-lapse video microscopy. We also discuss the relationship between chromosomal loss, cell proliferation, and the tumorigenic potential of p53-deficient cells lacking MdmX.

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. The late G1 rise in cyclin E/cdk2 kinase activity initiates centrosome duplication in mammalian cells and its activity is needed for centrosome duplication in Xenopus egg extracts. Since the half-time for cyclin E turnover is normally approximately 1 h in sea urchin zygotes, the different behaviors of centrosomes during G1 and S phase arrests could be due to differential losses of cyclin E and its associated kinase activities at these two arrest points. To better understand the mechanisms that limit centrosome duplication, we characterize the levels of cyclin E and its associated kinase activity at the S phase and G1 arrest points. We first demonstrate that cyclin E/cdk2 kinase activity is required for centrosome duplication and reduplication in sea urchin zygotes. Next we find that cyclin E levels and cyclin E/cdk2 kinase activities are both constitutively and equivalently elevated during both the S phase and G1 arrests. This indicates that centrosome duplication during the G1 arrest is limited by a block to reduplication under conditions permissive for duplication. The cytoplasmic conditions of S phase, however, abrogate this block to reduplication.