Jessica E. Hornick

Phosphorylation regulates targeting of cytoplasmic dynein to kinetochores during mitosis

Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis of mitotic dynein revealed a phosphorylation site in the dynein intermediate chains (ICs) that mediates binding to kinetochores. IC phosphorylation directs binding to zw10 rather than dynactin, and this interaction is needed for kinetochore dynein localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown to metaphase, but bioriented microtubule (MT) attachment and chromosome alignment induce IC dephosphorylation. IC dephosphorylation stimulates binding to dynactin and poleward streaming. MT depolymerization, release of kinetochore tension, and a PP1-γ mutant each inhibited IC dephosphorylation, leading to the retention of phosphodynein at kinetochores and reduced poleward streaming. The depletion of kinetochore dynactin by moderate levels of p50(dynamitin) expression disrupted the ability of dynein to remove checkpoint proteins by streaming at metaphase but not other aspects of kinetochore dynein activity. Together, these results suggest a new model for localization of kinetochore dynein and the contribution of kinetochore dynactin.

Chromosome cohesion decreases in human eggs with advanced maternal age.

Aneuploidy in human eggs increases with maternal age and can result in infertility, miscarriages, and birth defects. The molecular mechanisms leading to aneuploidy, however, are largely unknown especially in the human where eggs are exceedingly rare and precious. We obtained human eggs from subjects ranging from 16.4 to 49.7 years old following in vitro maturation of oocyte‐cumulus complexes isolated directly from surgically removed ovarian tissue. A subset of these eggs was used to investigate how age‐associated aneuploidy occurs in the human. The inter‐kinetochore distance between sister chromatids increased significantly with maternal age, indicating weakened cohesion. Moreover, we observed unpaired sister chromatids from females of advanced age. We conclude that loss of cohesion with increasing maternal age likely contributes to the well‐documented increased incidence of aneuploidy.

Isolated primate primordial follicles require a rigid physical environment to survive and grow in vitro

BACKGROUND In vitro follicle growth is a promising fertility preservation strategy in which ovarian follicles are cultured to produce mature and fertilization-competent oocytes. However, in primates, there has been limited success with in vitro follicle growth starting from primordial and primary follicles because adequate isolation methods and culture strategies have not been established. Understanding how to use primordial follicles for fertility preservation has significant implications because these follicles are the most abundant in the ovary, are found in all females and are fairly resistant to cryopreservation and chemotherapeutics. METHODS In the primate ovary, primordial follicles are concentrated near the collagen-rich ovarian cortex. To obtain these follicles, we separated the ovarian cortex prior to enzymatic digestion and enriched the primordial follicle concentration by using a novel double filtration system. To test the hypothesis that a rigid physical environment, as found in vivo, is optimal for survival, primordial follicles were cultured in different concentrations of alginate for up to 6 days. Follicle survival and morphology were monitored throughout the culture. RESULTS We found that primate ovarian tissue can be maintained for up to 24 h at 4°C without compromising tissue or follicle health. Hundreds of intact and viable primordial follicles were isolated from each ovary independent of animal age. Follicle survival and morphology were more optimal when follicles were cultured in 2% alginate compared with 0.5% alginate. CONCLUSIONS By mimicking the rigid ovarian environment through the use of biomaterials, we have established conditions that support primordial follicle culture. These results lay the foundations for studying the basic biology of primordial follicles in a controlled environment and for using primordial follicles for fertility preservation methods.

Toxoplasma gondii actively remodels the microtubule network in host cells

Toxoplasma gondii infection triggers host microtubule rearrangement and organelle recruitment around the parasite vacuole. Factors affecting initial stages of microtubule remodeling are unknown. To illuminate the mechanism, we tested the hypothesis that the parasite actively remodels host microtubules. Utilizing heat-killed parasites and time-lapse analysis, we determined microtubule rearrangement requires living parasites and is time dependent. We discovered a novel aster of microtubules (MTs) associates with the vacuole within 1h of infection. This aster lacks the concentrated foci of gamma (gamma)-tubulin normally associated with MT nucleation sites. Unexpectedly, vacuole enlargement does not correlate with an increase in MT staining around the vacuole. We conclude microtubule remodeling does not result from steric constraints. Using nocodazole washout studies, we demonstrate the vacuole nucleates host microtubule growth in-vivo via gamma-tubulin-associated sites. Moreover, superinfected host cells display multiple gamma-tubulin foci. Microtubule dynamics are critical for cell cycle control in uninfected cells. Using non-confluent monolayers, we show host cells commonly fail to finish cytokinesis resulting in larger, multinucleated cells. Our data suggest intimate interactions between T. gondii and host microtubules result in suppression of cell division and/or cause a mitotic defect, thus providing a larger space for parasite duplication.

Multiple follicle culture supports primary follicle growth through paracrine-acting signals

In vitro follicle growth in alginate hydrogels is a unique and versatile method for studying ovarian and follicle biology that may also have implications for fertility preservation. Current culture systems support the development of isolated mouse follicles from the secondary stage onward. However, it has been a challenge to grow smaller follicles in vitro due to the dissociation of the oocyte from companion somatic cells. Recent work has demonstrated that coculturing primary follicles with mouse embryonic fibroblasts or ovarian stromal cells supports follicle survival and growth. In this study, we demonstrate that follicles themselves can exert a beneficial coculture effect. When primary follicles were cultured in groups of five or ten (multiple follicle culture), there was increased growth and survival. The multiple follicle culture approach maintained follicle integrity and resulted in the formation of antral stage follicles containing meiotically competent gametes. The growth and survival of primary follicles were highly number dependent, with the most significant enhancement observed when the largest number of follicles was grown together. Our data suggest that the follicle unit is necessary to produce the secreted factors responsible for the supportive effects of multiple follicle culture, as neither denuded oocytes, oocyte-secreted factors, nor granulosa cells alone were sufficient to support early follicle growth in vitro. Therefore, there may be signaling from both the oocyte and the follicle that enhances growth but requires both components in a feedback mechanism. This work is consistent with current in vivo models for follicle growth and thus advances the movement to recapitulate the ovarian environment in vitro.

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin–independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.

Reproductive age-associated fibrosis in the stroma of the mammalian ovary.

Under normal physiological conditions, tissue remodeling in response to injury leads to tissue regeneration without permanent damage. However, if homeostasis between synthesis and degradation of extracellular matrix (ECM) components is altered, fibrosis - or the excess accumulation of ECM - can disrupt tissue architecture and function. Several organs, including the heart, lung and kidney, exhibit age-associated fibrosis. Here we investigated whether fibrosis underlies aging in the ovary - an organ that ages chronologically before other organs. We used Picrosirius Red (PSR), a connective tissue stain specific for collagen I and III fibers, to evaluate ovarian fibrosis. Using bright-field, epifluorescence, confocal and polarized light microscopy, we validated the specific staining of highly ordered PSR-stained fibers in the ovary. We next examined ovarian PSR staining in two mouse strains (CD1 and CB6F1) across an aging continuum and found that PSR staining was minimal in ovaries from reproductively young adult animals, increased in distinct foci in animals of mid-to-advanced reproductive age, and was prominent throughout the stroma of the oldest animals. Consistent with fibrosis, there was a reproductive age-associated increase in ovarian hydroxyproline content. We also observed a unique population of multinucleated macrophage giant cells, which are associated with chronic inflammation, within the ovarian stroma exclusively in reproductively old mice. In fact, several genes central to inflammation had significantly higher levels of expression in ovaries from reproductively old mice relative to young mice. These results establish fibrosis as an early hallmark of the aging ovarian stroma, and this altered microenvironment may contribute to the age-associated decline in gamete quality.

Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels

The in vitro growth of ovarian follicles is an emerging technology for fertility preservation. Various strategies support the culture of secondary and multilayer follicles from various species including mice, non‐human primate, and human; however, the culture of early stage (primary and primordial) follicles, which are more abundant in the ovary and survive cryopreservation, has been limited. Hydrogel‐encapsulating follicle culture systems that employed feeder cells, such as mouse embryonic fibroblasts (MEFs), stimulated the growth of primary follicles (70–80 µm); yet, survival was low and smaller follicles (<70 µm) rapidly lost structure and degenerated. These morphologic changes were associated with a breakdown of the follicular basement membrane; hence, this study investigated ascorbic acid based on its role in extracellular matrix (ECM) deposition/remodeling for other applications. The selection of ascorbic acid was further supported by a microarray analysis that suggested a decrease in mRNA levels of enzymes within the ascorbate pathway between primordial, primary, and secondary follicles. The supplementation of ascorbic acid (50 µg/mL) significantly enhanced the survival of primary follicles (<80 µm) cultured in alginate hydrogels, which coincided with improved structural integrity. Follicles developed antral cavities and increased to diameters exceeding 250 µm. Consistent with improved structural integrity, the gene/protein expression of ECM and cell adhesion molecules was significantly changed. This research supports the notion that modifying the culture environment (medium components) can substantially enhance the survival and growth of early stage follicles. Biotechnol. Bioeng. 2014;111: 1417–1429.

Kinesins to the core: The role of microtubule-based motor proteins in building the mitotic spindle midzone

In mammalian cultured cells the initiation of cytokinesis is regulated - both temporally and spatially - by the overlapping, anti-parallel microtubules of the spindle midzone. This region recruits several key central spindle components: PRC-1, polo-like kinase 1 (Plk-1), the centralspindlin complex, and the chromosome passenger complex (CPC), which together serve to stabilize the microtubule overlap, and also to coordinate the assembly of the cortical actin/myosin cytoskeleton necessary to physically cleave the cell in two. The localization of these crucial elements to the spindle midzone requires members of the kinesin superfamily of microtubule-based motor proteins. Here we focus on reviewing the role played by a variety of kinesins in both building and operating the spindle midzone machinery during cytokinesis.

Centrosome Biogenesis Continues in the Absence of Microtubules During Prolonged S-Phase Arrest

When CHO cells are arrested in S‐phase, they undergo repeated rounds of centrosome duplication without cell‐cycle progression. While the increase is slow and asynchronous, the number of centrosomes in these cells does rise with time. To investigate mechanisms controlling this duplication, we have arrested CHO cells in S‐phase for up to 72 h, and coordinately inhibited new centriole formation by treatment with the microtubule poison colcemid. We find that in such cells, the pre‐existing centrosomes remain, and a variable number of foci—containing α/γ‐tubulin and centrin 2—assemble at the nuclear periphery. When the colcemid is washed out, the nuclear‐associated foci disappear, and cells assemble new centriole‐containing centrosomes, which accumulate the centriole scaffold protein SAS‐6, nucleate microtubule asters, and form functional mitotic spindle poles. The number of centrosomes that assemble following colcemid washout increases with duration of S‐phase arrest, even though the number of nuclear‐associated foci or pre‐existing centrosomes does not increase. This suggests that during S‐phase, a cryptic generative event occurs repeatedly, even in the absence of new triplet microtubule assembly. When triplet microtubule assembly is restored, these cryptic generative events become realized, and multiple centriole‐containing centrosomes assemble. J. Cell. Physiol. 225: 454–465, 2010.