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[
International Worm Meeting,
2009]
Cells, like whole organisms, have an incredible diversity of form. Cell patterning is achieved through the translation of polarizing cues into the specific arrangement of organelles and subcellular structures. Chief among these is the centrosome, the major microtubule organizing center of the cell. Despite being named for its central location in the cell, the centrosome can often occupy asymmetrical positions in differentiated cells, where it can contribute to the formation or function of cell structures such as cilia. During cell state transitions such as wound healing and polarization, centrosomes can also undergo transient repositioning within cells, and the function of this repositioning is not known. In the development of the C. elegans intestinal epithelia, centrosomes transiently shift from an anterior or posterior position in the cell to an orthogonal position at the future apical surface. This repositioning occurs at a developmental stage when cells are just beginning to form a polarized epithelium, and lack hallmarks such as apical junctions. We find that, following repositioning, centrosomal proteins become deposited along the apical surface of intestinal cells, suggesting that one function of centrosome repositioning might be to shuttle nucleators of microtubule assembly to the apical surface prior to polarization. We show that the PAR-3/PAR-6/PKC-3 complex localizes apically as has been shown for other epithelia and are exploring the role of the centrosome in this localization. Finally, we are using visual genetic screens to identify genes that are involved in centrosome positioning in epithelial cells, and that link centrosomes to cell polarity.
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[
International Worm Meeting,
2013]
The centrosome is the major microtubule organizing center (MTOC) in dividing cells. In many types of differentiated cells, however, MTOC function is reassigned to non-centrosomal sites. We are using C. elegans intestinal cells to analyze how MTOC function is reassigned to the apical surface of epithelial cells. After the terminal cell divisions, the centrosomes of intestinal cells move near the future apical membranes, and the post-mitotic centrosomes lose all, or most, of their associated microtubules. We show that microtubule-nucleating proteins such as g-tubulin that are centrosome components in dividing cells become localized to the apical membrane, which becomes highly enriched in microtubules. Our results suggest that centrosomes are critical to specify the apical membrane as the new MTOC. First, g-tubulin fails to accumulate apically in wild-type cells following laser ablation of the centrosome. Second, g-tubulin appears to redistribute directly from the migrating centrosome, forming a nascent MTOC at the lateral membrane before redistributing apically. Electron microscopy of embryos staged at the transition between lateral and apical g-tubulin show electron dense material associated with small clusters of microtubules at both lateral and apical sites. These data suggest that the reassignment of MTOC function from centrosomes to the apical membrane is associated with a physical hand-off of nucleators of microtubule assembly.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Cell patterning is achieved through the translation of polarizing cues into the specific arrangement of organelles and subcellular structures. Chief among these is the centrosome, the major microtubule organizing center of the cell. During cell state transitions such as wound healing and polarization, centrosomes reposition within cells, and the function of this repositioning is not known. In the development of the C. elegans intestinal epithelia, centrosomes shift from an anterior or posterior position in cells following the final round of division to an orthogonal position at the future apical surface. This repositioning occurs at a developmental stage when cells are just beginning to form a polarized epithelium, and lack hallmarks such as apical junctions. We are interested in learning if and how centrosome localization influences cell polarization. We find that, prior to centrosome repositioning, polarity proteins such as PAR-6 and PAR-3 accumulate in foci at anterior or posterior membranes, and that these foci traffic with centrosomes to the future apical surface. PAR-6 is not required for centrosome repositioning; we are currently testing the role of PAR-3 in this process. Microtubules appear to be required for the initiation and progression of centrosome repositioning. This requirement may be mediated through dynein as maternal and zygotic depletion of the dynein accessory protein roadblock results in centrosome positioning defects. Following repositioning, centrosomal proteins such as ?-tubulin become deposited along the apical surface of intestinal cells, suggesting that one function of centrosome repositioning might be to shuttle nucleators of microtubule assembly to the apical surface prior to polarization.
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[
International Worm Meeting,
2019]
Dividing and differentiating cells require different arrangements of microtubules to function. Mitotic cells establish centrosomes as microtubule organizing centers (MTOCs), producing radial microtubule arrays that are critical for chromosome segregation. In contrast, polarized epithelial cells form parallel arrays of microtubules emanating from a non-centrosomal MTOC, the apical membrane, that promote cell polarity and intracellular transport. During development and tissue homeostasis, some polarized epithelial cells divide, presenting an important but poorly understood obstacle: microtubules must temporarily cycle between the apical surface and the centrosomes. The developing C. elegans intestine provides an excellent in vivo epithelial model to study how this microtubule reorganization is achieved. After the 16-cell embryonic intestine polarizes and establishes an apical MTOC, exactly four "E16*" cells divide again. The E16* divisions involve a rapid change in microtubule organization from apical to centrosomal as cells enter mitosis, and back to apical upon mitotic exit (Yang and Feldman 2015). Using fluorescent markers, genetic screens, and tissue-specific protein depletion with live imaging, we are testing the hypothesis that apical polarity proteins control microtubule reorganization during the E16* divisions. During mitosis, we observe that like microtubules, MTOC-associated proteins also leave the apical membrane as the centrosome becomes the MTOC. However, the apical PAR polarity proteins remain at the apical membrane during the E16* divisions, suggesting that they may act as a memory mark and help direct the return of microtubules and MTOC proteins after division. Consistent with this model, we have found that intestine-specific depletion of the apical polarity proteins PAR-6 and PKC-3 disrupts MTOC reformation following the E16* division. A pilot forward genetic suppressor screen has isolated a suppressor of the MTOC defects caused by PAR-6 depletion. These experiments reveal a role for PAR proteins in returning MTOC function to the apical membrane following mitosis, a critical step in epithelial cell divisions across organisms.
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[
International Worm Meeting,
2021]
Polyploidy, the condition in which cells obtain additional sets of chromosomes, is a normal feature of cells in many diploid organisms, yet little is known about why an increase in ploidy may be advantageous. Polyploidy can protect liver cells from becoming cancerous after the loss of a tumor suppressor gene, but this cannot be the only purpose as many cell types become polyploid through post-mitotic DNA replication. A second hypothesis is that the increased DNA content in polyploid cells supports greater cell size. For example, the growth of the nematode C. elegans is driven primarily through cell growth as opposed to cell proliferation, and its overall body size is thought to be regulated by the nuclear ploidy of its syncytial epidermis. The C. elegans intestine is also composed of large, polyploid cells, but it is unknown whether any physiological benefits result from this increased DNA content. Using a tissue- and temporally-specific protein degradation system, we degraded a C. elegans G1/S cyclin dependent kinase, CDK-2, to reduce the ploidy of the intestine and the epidermis. Preliminary results show that body size is not reduced upon reduction of epidermal polyploidy. In contrast, ploidy reduction in the intestine leads to slow growth, small body size, and reduced lifespan. Finally, low ploidy intestines may not have wild-type capacity for nutrient absorption, as these worms show signs of starvation even with abundantly available food. These results implicate polyploidy as essential for the function of the intestine and challenge existing models of C. elegans growth regulation. Future studies will investigate whether polyploidy is involved in other intestinal processes such as yolk production and innate immunity.
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[
International Worm Meeting,
2021]
Epithelial cells are specialized to protect our bodies and tissues and interface with the outside environment. During both development and malignant transformation into carcinomas, a crucial yet poorly understood facet of epithelial cell biology is how different epithelial tissues connect to and align with their neighboring epithelial tissues. For circulating cancer cells, productive attachment to foreign tissues is a rate-limiting step in metastasis formation. For the developing luminal epithelia that line internal organs, forming an epithelial bridge to the epithelia on the outside of the body is necessary for viability. In particular, alignment of the apicobasal axis and cell-cell adhesion complexes between neighboring cells is essential. The development of the C. elegans digestive tract involves the formation of such epithelial bridges, including the two rectal valve cells that bridge between the intestinal and rectal cells that derive from two different lineages (E and ABp). This connection between the intestine and the rectum completes a toroid of epithelial cells that encases and protects the worm. Despite their importance in forming the connection between the developing intestine and the invaginating rectal epithelium, little is known about the cellular organization and morphogenesis of the rectal valve cells. My preliminary work suggests that the polarization of apicobasal proteins within the valve cells may be key to forming this epithelial bridge. During their morphogenesis, valve cells possess one anterior puncta and one posterior puncta of conserved apical proteins, which were previously unable to be visualized separately from surrounding intestinal and rectal signal. Ablation of the posterior intestinal cells disrupts the formation of the anterior valve cell apical puncta. In addition, the activity of the conserved adhesion protein HMR-1/E-cadherin, but not components of the apical PAR complex, specifically within the posterior intestinal cells is required to form the anterior valve cell apical puncta. I am further exploring the hypothesis that intestinal adhesion complexes instruct valve cell polarization. In addition, I am currently generating tools to enable better cell-type specific protein depletions within the intestine and different populations of rectal cells with the aim of testing how polarity and adhesion complexes within these cells affect their epithelial neighbors. Together, these data will improve our understanding of epithelial cell organization in different in vivo contexts ranging from morphogenesis to tubule formation, from metastatic colonization to wound healing. How spatial information derived from the polarization of one epithelia tissue is able to instruct the polarization of another, neighboring epithelial tissue has broad implications for explaining how epithelial neighbors meet and align to form functional organ systems.
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[
International Worm Meeting,
2021]
The ability of epithelia to protect and line our organs and bodies requires continued epithelial integrity through assaults like cell division and growth. Epithelia are made of adherent cells that polarize along an apicobasal axis and act as selective barriers; the apical surfaces of cells are collectively oriented toward the lumen or exterior and are separated from basolateral domains by junctional complexes that adhere neighboring epithelial cells. Tissue integrity, or correct and continued cell polarity and adhesion, is essential for epithelial function, but during development, epithelia face assaults on their integrity, such as cells dividing or resizing their apical surfaces. The developing C. elegans intestine provides a simple in vivo model to study how epithelia overcome these assaults. Intestinal cells polarize with apical surfaces facing a central midline, the future lumen. Four cells divide again, and all cells elongate to build a continuous apical surface and lumen and produce a functional intestinal tube. To understand what happens to polarized features during epithelial cell division, we live imaged cytoskeletal, polarity, and junctional proteins in mitotic intestinal cells. We observed that ACT-5/actin, apical PAR proteins, and the junctional proteins HMR-1/E-cadherin and DLG-1/Discs large remained localized during mitosis; in contrast, apical microtubules and associated proteins were transiently lost during mitosis and returned to the apical surface following mitotic exit. This loss of apical microtubules appeared to be coupled to the building of the microtubule-based mitotic spindle, suggesting a functional switch between these structures. Based on our localization findings, we hypothesized that PAR proteins actively maintain apical identity and continuity during epithelial assaults, directing the return of apical microtubules after mitosis and expanding the apical surface during elongation. Using intestine-specific depletion of PAR-6/Par6 and PKC-3/aPKC, we found that apical microtubule organization was indeed disrupted following mitosis and that gaps in apical and junctional proteins formed between cells both following mitosis and over time as intestinal cells elongated. When we examined the resulting PAR-depleted larval intestines, we found that gaps in apical and junctional proteins were present and correlated with luminal constrictions. The consequences of these intestinal defects were larvae that arrested and died with edematous intestines that failed to pass food. These experiments reveal a role for PAR proteins in maintaining apical and junctional continuity through mitosis and elongation, a critical feature of epithelial integrity across organisms. NIH NIGMS K99
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
During morphogenesis of the pharynx and the valve, cells within a common epithelial primordium develop specialized shapes that determine organ morphology and function. We are studying how two of these cells, the pharyngeal cell
pm8 and the valve cell
vpi1, become adjacent single-cell tubes.
pm8 and
vpi1 are born on the dorsal half of the pharyngeal/valve primordium. They subsequently invade between ventral cells, wrap around the midline and self-fuse. In part, these events require Notch signaling in
pm8 that directly regulates expression of several target genes. Morphogenesis of
pm8 and
vpi1 allows a thick basement membrane to form between these single-cell tubes, effectively compartmentalizing the pharynx from the valve. We previously showed that the alpha-integrin
ina-1 is required to compartmentalize the pharynx; in
ina-1 mutant s,
pm8 can be located ectopically between valve cells, or even intestinal cells [1]. Here, we conducted a pilot genetic screen to identify mutants with similar compartmentalization defects, and cloned two of these. The first,
zu470, is a new, nonsense mutation (W610>*) in
ina-1 that was not analyzed further. The second,
zu471, is a missense mutation (R489>H) in
egl-43.
egl-43 encodes a zinc-finger transcription factor homologous to the mammalian oncogene EVI-1 [2]. We found that
egl-43 mutants are defective in remodeling the apical junctions of
pm8 and
vpi1 during morphogenesis, and fail to form the basement membrane between the pharynx and the valve. We found that EGL-43 is expressed in
vpi1 during morphogenesis, but shows no detectable expression in
pm8. Surprisingly, however,
egl-43 mutants show variable defects in Notch-dependent gene expression in
pm8. Thus,
egl-43 may have a pre-morphogenesis role in regulating gene expression in
pm8 precursors (MSaaap lineage) and/or in the precursors of the Notch ligand-expressing cells (MSaapa lineage). [1] Rasmussen et al. Dev Cell 14, 559 (2008). [2] Garriga et al. Genes Dev 7, 2097 (1993).
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[
International Worm Meeting,
2021]
During development, the centrosome acts as a microtubule organizing center (MTOC) in mitotic cells, forming radial arrays essential to separate cellular components between daughter cells. Microtubules are organized at the centrosome by pericentriolar material (PCM) complexes. After mitosis, during cell differentiation the fate of the centrosome is going to be diverse depending of cell fate. In differentiated epithelial cells, the centrosome is inactivated, losing its PCM and having the MTOC function redirected to other cellular components, in contrast, other ciliated cells have the centrosome and its centrioles repurposed into basal bodies to ciliary structures. We are using C. elegans as a model to characterize MTOC recruitment and regulation at the centrosome. In C. elegans, the PCM is organized around the centrioles in a partial concentric overlay of protein spheres, in which the two main scaffolding protein, SPD-2/CEP192 and SPD-5 which localize the microtubule nucleating complex -TuRC to the centrosome, partially overlap delimiting two main body - an inner sphere with both partner and an outer sphere with SPD-5 (Magescas et al 2019). Upon differentiation centrosome lose their PCM, leaving 'naked' centrioles, like in intestinal cells. Interestingly, analysis of SPD-5 and -TuRC proteins revealed that ciliated sensory neurons, SPD-5 and most MTOC proteins remains at the ciliary base while centriolar protein and SPD-2 are lost. Those complexes organize the MTOC function at the base of cilia and are critical for ciliogenesis, as depletion of SPD-5 produces aberrant cilia. Interestingly, contrary to the current model, similar loss of SPD-2 in cycling intestinal cells prior to differentiation doesn't result in the loss of SPD-5 at the centrosome, nor it impairs centrosomal function. Based on our data we propose that the PCM is composed of different subcomplexes revolving around SPD-5 that are differently regulated, working in parallel to drive the MTOC function.
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[
International Worm Meeting,
2017]
Microtubule organization is critical for cell function. Nearly all dividing animal cells use the centrosome as a microtubule organizing center (MTOC), where microtubules tether chromosomes to the spindle poles to facilitate the correct segregation of DNA between daughter cells. By contrast, differentiated cells organize their microtubules in a wide variety of patterns, and establish specific noncentrosomal locations as MTOCs to achieve these microtubule arrangements. While much is known about how centrosomes organize microtubules, little is known about the composition of noncentrosomal MTOCs (ncMTOCs), how these sites are designated, or how they organize microtubules. A simple hypothesis is that an ncMTOC is essentially the MTOC features of a centrosome targeted to a different cellular location. Our studies suggest that the ncMTOC that forms at the apical surface of the polarized C. elegans intestine is in fact different in its composition and protein requirements for microtubule organization than the centrosome. We have examined the localization of several CRISPR-tagged microtubule- and MTOC-associated proteins in the intestinal ncMTOC, and observe three classes of proteins: (1) only at the centrosome (SPD-2/Cep192, SPD-5), (2) only at the ncMTOC (PTRN-1/CAMSAP, NOCA-1/Ninein), and (3) at whichever location is the active MTOC (?-TuRC proteins, AIR-1/Aurora A). To test the requirement of several factors in intestinal ncMTOCs, including AIR-1 and the ?-TuRC components GIP-1 and Mozart, we have optimized the ZIF-1/ZF protein degradation system to allow for the removal of early essential proteins from tissues of interest. We find evidence that the microtubule nucleation complex ?-TuRC must be intact for the recruitment of ?-TuRC components to the ncMTOC. Surprisingly, microtubules are still made and correctly localized to the apical ncMTOC even when ?-TuRC and AIR-1 are compromised, and we are currently analyzing the dynamics of these microtubules. These results suggest that differentiated cells use novel mechanisms to create a functional ncMTOC that organizes their microtubules.