Jessica L. Feldman

A Role for the Centrosome and PAR-3 in the Hand-Off of MTOC Function during Epithelial Polarization

BACKGROUND The centrosome is the major microtubule organizing center (MTOC) in dividing cells and in many postmitotic, differentiated cells. In other cell types, however, MTOC function is reassigned from the centrosome to noncentrosomal sites. Here, we analyze how MTOC function is reassigned to the apical membrane of C. elegans intestinal cells. RESULTS After the terminal intestinal cell division, the centrosomes and nuclei move near the future apical membranes, and the postmitotic centrosomes lose all, or nearly all, of their associated microtubules. We show that microtubule-nucleating proteins such as γ-tubulin and CeGrip-1 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, γ-tubulin appears to redistribute directly from the migrating centrosome onto the lateral then apical membrane. Second, γ-tubulin fails to accumulate apically in wild-type cells following laser ablation of the centrosome. We show that centrosomes localize apically by first moving toward lateral foci of the conserved polarity proteins PAR-3 and PAR-6 and then move together with these foci toward the future apical surface. Embryos lacking PAR-3 fail to localize their centrosomes apically and have aberrant localization of γ-tubulin and CeGrip-1. CONCLUSIONS These data suggest that PAR proteins contribute to apical polarity in part by determining centrosome position and that the reassignment of MTOC function from centrosomes to the apical membrane is associated with a physical hand-off of nucleators of microtubule assembly.

Katanin Knockdown Supports a Role for Microtubule Severing in Release of Basal Bodies before Mitosis in Chlamydomonas

Katanin is a microtubule-severing protein that participates in the regulation of cell cycle progression and in ciliary disassembly, but its precise role is not known for either activity. Our data suggest that in Chlamydomonas, katanin severs doublet microtubules at the proximal end of the flagellar transition zone, allowing disengagement of the basal body from the flagellum before mitosis. Using an RNA interference approach we have discovered that severe knockdown of the p60 subunit of katanin, KAT1, is achieved only in cells that also carry secondary mutations that disrupt ciliogenesis. Importantly, we observed that cells in the process of cell cycle-induced flagellar resorption sever the flagella from the basal bodies before resorption is complete, and we find that this process is defective in KAT1 knockdown cells.

Microtubule-organizing centers: from the centrosome to non-centrosomal sites.

The process of cellular differentiation requires the distinct spatial organization of the microtubule cytoskeleton, the arrangement of which is specific to cell type. Microtubule patterning does not occur randomly, but is imparted by distinct subcellular sites called microtubule-organizing centers (MTOCs). Since the discovery of MTOCs fifty years ago, their study has largely focused on the centrosome. All animal cells use centrosomes as MTOCs during mitosis. However in many differentiated cells, MTOC function is reassigned to non-centrosomal sites to generate non-radial microtubule organization better suited for new cell functions, such as mechanical support or intracellular transport. Here, we review the current understanding of non-centrosomal MTOCs (ncMTOCs) and the mechanisms by which they form in differentiating animal cells.

C. elegans Germ Cells Show Temperature and Age-Dependent Expression of Cer1, a Gypsy/Ty3-Related Retrotransposon

Virus-like particles (VLPs) have not been observed in Caenorhabditis germ cells, although nematode genomes contain low numbers of retrotransposon and retroviral sequences. We used electron microscopy to search for VLPs in various wild strains of Caenorhabditis, and observed very rare candidate VLPs in some strains, including the standard laboratory strain of C. elegans, N2. We identified the N2 VLPs as capsids produced by Cer1, a retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons. Cer1 expression is age and temperature dependent, with abundant expression at 15°C and no detectable expression at 25°C, explaining how VLPs escaped detection in previous studies. Similar age and temperature-dependent expression of Cer1 retrotransposons was observed for several other wild strains, indicating that these properties are common, if not integral, features of this retroelement. Retrotransposons, in contrast to DNA transposons, have a cytoplasmic stage in replication, and those that infect non-dividing cells must pass their genomic material through nuclear pores. In most C. elegans germ cells, nuclear pores are largely covered by germline-specific organelles called P granules. Our results suggest that Cer1 capsids target meiotic germ cells exiting pachytene, when free nuclear pores are added to the nuclear envelope and existing P granules begin to be removed. In pachytene germ cells, Cer1 capsids concentrate away from nuclei on a subset of microtubules that are exceptionally resistant to microtubule inhibitors; the capsids can aggregate these stable microtubules in older adults, which exhibit a temperature-dependent decrease in egg viability. When germ cells exit pachytene, the stable microtubules disappear and capsids redistribute close to nuclei that have P granule-free nuclear pores. This redistribution is microtubule dependent, suggesting that capsids that are released from stable microtubules transfer onto new, dynamic microtubules to track toward nuclei. These studies introduce C. elegans as a model to study the interplay between retroelements and germ cell biology.

ASQ2 Encodes a TBCC-like Protein Required for Mother-Daughter Centriole Linkage and Mitotic Spindle Orientation

An intriguing feature of centrioles is that these highly complicated microtubule-based structures duplicate once per cell cycle, affording the cell precise control over their number. Each cell contains exactly two centrioles, linked together as a pair, one of which is a mother centriole formed in a previous cell cycle and the other of which is a daughter centriole whose assembly is templated by the mother. Neither the molecular basis nor the functional role of mother-daughter centriole linkage is understood. We have identified a mutant, asq2, with defects in centriole linkage. asq2 mutant cells have variable numbers of centrioles and centriole positioning defects. Here, we show that ASQ2 encodes the conserved protein Tbccd1, a member of a protein family including a tubulin folding cochaperone and the retinitis pigmentosa protein RP2, involved in tubulin quality control during ciliogenesis. We characterize mitosis in asq2 cells and show that the majority of cells establish a bipolar spindle but have defects in spindle orientation. Few asq2 cells have centrioles at both poles, and these cells have properly positioned spindles, indicating that centrioles at the poles might be important for spindle orientation. The defects in centriole number control, centriole positioning, and spindle orientation appear to arise from perturbation of centriole linkage mediated by Tbccd1/Asq2p.

SPD-2/CEP192 and CDK Are Limiting for Microtubule-Organizing Center Function at the Centrosome

The centrosome acts as the microtubule-organizing center (MTOC) during mitosis in animal cells. Microtubules are nucleated and anchored by γ-tubulin ring complexes (γ-TuRCs) embedded within the centrosomes pericentriolar material (PCM). The PCM is required for the localization of γ-TuRCs, and both are steadily recruited to the centrosome, culminating in a peak in MTOC function in metaphase. In differentiated cells, the centrosome is often attenuated as an MTOC and MTOC function is reassigned to non-centrosomal sites such as the apical membrane in epithelial cells, the nuclear envelope in skeletal muscle, and down the lengths of axons and dendrites in neurons. Hyperactive MTOC function at the centrosome is associated with epithelial cancers and with invasive behavior in tumor cells. Little is known about the mechanisms that limit MTOC activation at the centrosome. Here, we find that MTOC function at the centrosome is completely inactivated during cell differentiation in C. elegans embryonic intestinal cells and MTOC function is reassigned to the apical membrane. In cells that divide after differentiation, the cellular MTOC state switches between the membrane and the centrosome. Using cell fusion experiments in live embryos, we find that the centrosome MTOC state is dominant and that the inactive MTOC state of the centrosome is malleable; fusion of a mitotic cell to a differentiated or interphase cell results in rapid reactivation of the centrosome MTOC. We show that conversion of MTOC state involves the conserved centrosome protein SPD-2/CEP192 and CDK activity from the mitotic cell.

A cell-based screen for inhibitors of flagella-driven motility in Chlamydomonas reveals a novel modulator of ciliary length and retrograde actin flow†

Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical‐biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time‐scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose‐dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound “ciliabrevin” due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose‐dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug.

PCR-based assay for mating type and diploidy in Chlamydomonas.

Characterizing Chlamydomonas The unicellular green alga, Chlamydomonas reinhardtii, is a powerful genetic model for the analysis of a variety of cellular functions. Critical assays for genetic anal...

The kinase regulator mob1 acts as a patterning protein for stentor morphogenesis.

Here we demonstrate that RNAi can be used in molecular studies of the giant single-celled ciliate Stentor coeruleus, revealing morphogenetic functions of Mob1 and highlighting the potential of this classical model for studies of morphogenesis and regeneration.

Cell Interactions and Patterned Intercalations Shape and Link Epithelial Tubes in C. elegans

Many animal organs are composed largely or entirely of polarized epithelial tubes, and the formation of complex organ systems, such as the digestive or vascular systems, requires that separate tubes link with a common polarity. The Caenorhabditis elegans digestive tract consists primarily of three interconnected tubes—the pharynx, valve, and intestine—and provides a simple model for understanding the cellular and molecular mechanisms used to form and connect epithelial tubes. Here, we use live imaging and 3D reconstructions of developing cells to examine tube formation. The three tubes develop from a pharynx/valve primordium and a separate intestine primordium. Cells in the pharynx/valve primordium polarize and become wedge-shaped, transforming the primordium into a cylindrical cyst centered on the future lumenal axis. For continuity of the digestive tract, valve cells must have the same, radial axis of apicobasal polarity as adjacent intestinal cells. We show that intestinal cells contribute to valve cell polarity by restricting the distribution of a polarizing cue, laminin. After developing apicobasal polarity, many pharyngeal and valve cells appear to explore their neighborhoods through lateral, actin-rich lamellipodia. For a subset of cells, these lamellipodia precede more extensive intercalations that create the valve. Formation of the valve tube begins when two valve cells become embedded at the left-right boundary of the intestinal primordium. Other valve cells organize symmetrically around these two cells, and wrap partially or completely around the orthogonal, lumenal axis, thus extruding a small valve tube from the larger cyst. We show that the transcription factors DIE-1 and EGL-43/EVI1 regulate cell intercalations and cell fates during valve formation, and that the Notch pathway is required to establish the proper boundary between the pharyngeal and valve tubes.