Gotta M et al. (2001) Nat Cell Biol "Distinct roles for Galpha and Gbetagamma in regulating spindle position and orientation in Caenorhabditis elegans embryos."

Ahringer JA, Gotta M

[Nat Cell Biol, 2001]

Correct placement and orientation of the mitotic spindle is essential for segregation of localized components and positioning of daughter cells. Although these processes are important in many cells, few factors that regulate spindle placement are known. Previous work has shown that GPB-1, the Gbeta subunit of a heterotrimeric G protein, is required for orientation of early cell division axes in C. elegans embryos. Here we show that GOA-1 (a Galphao) and the related GPA-16 are the functionally redundant Galpha subunits and that GPC-2 is the relevant Ggamma subunit that is required for spindle orientation in the early embryo. We show that Galpha and Gbetagamma are involved in controlling distinct microtubule-dependent processes. Gbetagamma is important in regulating migration of the centrosome around the nucleus and hence in orientating the mitotic spindle. Galpha is required for asymmetric spindle positioning in the one-celled embryo.

Gotta M et al. (2001) Curr Biol "CDC-42 controls early cell polarity and spindle orientation in C. elegans."

Ahringer J, Abraham MC, Gotta M

[Curr Biol, 2001]

Background: Generation of asymmetry in the one-cell embryo of C. elegans establishes the anterior-posterior axis (A-P), and is necessary far the proper identity of early blastomeres. Conserved PAR proteins are asymmetrically distributed and are required for the generation of this early asymmetry. The small G protein Cdc42 is a key regulator of polarity in other systems, and recently it has been shown to interact with the mammalian homolog of PAR-6. The function of Cdc42 in C elegans had not yet been investigated, however. Results: Here, we show that C. elegans cdc-42 plays an essential role in the polarity of the one-cell embryo and the proper localization of PAR proteins. Inhibition of cdc-42 using RNA interference results in embryos with a phenotype that is nearly identical to par-3, par-6, and pkc-3 mutants, and asymmetric localization of these and other PAR proteins is lost. We further show that C. elegans CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs. Conclusions: Our results show that CDC-42 acts in concert with the PAR proteins to control the polarity of the C. elegans embryo, and provide evidence that the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit.

Gotta M et al. (2001) Curr Opin Genet Dev "Axis determination in C. elegans: initiating and transducing polarity."

Gotta M, Ahringer J

[Curr Opin Genet Dev, 2001]

The anterior-posterior axis in Caenorhabditis elegans is determined by the sperm and leads to the asymmetric localisation of PAR (partitioning-defective) proteins, which are critical for polarity. New findings demonstrate that sperm asters play a critical role and suggest models for how PAR asymmetry is established. In addition, studies of blastomere fate determination and heterotrimeric G proteins have started to uncover how initial polarity may be translated into the asymmetric distribution of maternal proteins and the control of spindle position.

Gotta M et al. (2003) Curr Biol "Asymmetrically distributed C. elegans homologs of AGS3/PINS control spindle position in the early embryo."

Lanier SM, Gotta M, Dong Y, Ahringer J, Peterson YK

[Curr Biol, 2003]

Background: Spindle positioning during an asymmetric cell division is of fundamental importance to ensure correct size of daughter cells and segregation of determinants. In the C. elegans embryo, the first spindle is asymmetrically positioned, and this asymmetry is controlled redundantly by two heterotrimeric Galpha subunits, GOA-1 and GPA-16. The Galpha subunits act downstream of the PAR polarity proteins, which control the relative pulling forces acting on the poles. How these heterotrimeric G proteins are regulated and how they control spindle position is still unknown. Results: Here we show that the Ga subunits are regulated by a receptor-independent mechanism. RNAi depletion of gpr-1 and gpr-2, homologs of mammalian AGS3 and Drosophila PINS (receptor-independent G protein regulators), results in a phenotype identical to that of embryos depleted of both GPA-16 and GOA-1; the first cleavage is symmetric, but polarity is not affected. The loss of spindle asymmetry after RNAi of gpr-1 and gpr-2 appears to be the result of weakened pulling forces acting on the poles. The GPR protein(s) localize around the cortex of one-cell embryos and are enriched at the posterior. Thus, asymmetric G protein regulation could explain the posterior displacement of the spindle. Posterior enrichment is abolished in the absence of the PAR polarity proteins PAR-2 or PAR-3. In addition, LIN-5, a coiled-coil protein also required for spindle positioning, binds to and is required for cortical association of the GPR protein(s). Finally, we show that the GPR domain of GPR-1 and GPR-2 behaves as a GDP dissociation inhibitor for GOA-1, and its activity is thus similar to that of mammalian AGS3. Conclusions: Our results suggest that GPR-1 and/or GPR-2 control an asymmetry in forces exerted on the spindle poles by asymmetrically modulating the activity of the heterotrimeric G protein in response to a signal from the

M Gotta et al. (1999) International C. elegans Meeting "Control of cell division axis choice in C.elegans"

M Gotta, J Ahringer

[International C. elegans Meeting, 1999]

The orientation of the cleavage plane is a critical aspect of development. The cleavage plane is established by the position and orientation of the mitotic spindle, which is set by the position of the centrosomes. In C. elegans embryos, cell division planes follow a fixed pattern, allowing abnormalities to be easily analysed. In previous work, we found that gpb-1 , which encodes the beta subunit of a heterotrimeric GTP-binding protein, has a crucial role in spindle orientation. In embryos lacking GPB-1 activity, mitotic spindles in early cell divisions are formed properly but they are randomly oriented instead of following a fixed pattern. Lack of GPB-1 does not appear to affect the polarity of the zygote. Interestingly, GPB-1 localises to the asters during cell division. To further characterise the G protein involved in spindle orientation, we sought the G g partner. There are two G g genes in the genome, gpc-1 and gpc-2 . RNA interference to gpc-2 , but not to gpc-1 , causes embryo lethality. Staining of early embryos with anti-tubulin antibodies shows that cleavage planes in early cell divisions are abnormal. However, the first cleavage is asymmetric and P granules are correctly localised to the germline precursor cells. This suggests that GPC-2 is probably the partner of GPB-1 in controlling spindle orientation. We are currently trying to find interactors of G bg by a three hybrid screen. As a second approach, we are trying to identify other factors involved in spindle orientation by RNAi to candidate genes based on the homology with yeast polarisation during mating which is controlled by a heterotrimeric G protein. Upon activation by the receptor, the G bg dimer activates downstream factors such as Cdc42p and Ste20p. Injection of dsRNA for two of the many Ste20p homologues show no phenotype, although this may reflect a redundant function. RNAi to the C. elegans Cdc42p homologue causes embryo lethality and the early embryos are osmotically sensitive. In these embryos cleavage planes are abnormally oriented. In addition the first cleavage can be symmetric and P granules are not properly localised. We have undertaken a two hybrid screen with CDC-42 and we are currently analysing the interacting clones by RNAi.

Calvi, I. et al. (2019) International Worm Meeting "Cell polarity regulation by the mitotic kinase PLK-1 in C. elegans embryos."

Gotta, M., Calvi, I.

[International Worm Meeting, 2019]

Cell polarity is a fundamental property of cells and it is crucial for many biological processes at the cellular and organismal level. The one-cell C. elegans embryo is polarized along the anterior-posterior axis and this mechanism is essential for asymmetric cell division, a specialized division that results in two cells with a different fate. PAR proteins, a network of highly dynamic proteins with scaffold and/or enzyme function, mediate and control cell polarization in the one-cell C. elegans embryos. They form two distinct cortical domains, the anterior and the posterior PAR domains. The formation of these domains takes place in two distinct phases: the polarity establishment and maintenance phases (reviewed in Rose and Gonczy, 2014). Just after the fertilization, the anterior PAR proteins are uniformly distributed at the cortex. During polarity establishment, PAR-3, an anterior PAR protein, oligomerizes and forms big clusters (Dickinson et al., 2017). Formation of these clusters is required for polarity establishment as it couples PAR-3 itself and the other anterior PAR proteins, PAR-6 and aPKC, to the cortical flows, therefore enabling movement to the anterior. After pronuclear meeting, PAR-3 clusters dissolve and the maintenance phase begins. We found that phosphorylation of PAR-3 by PLK-1 inhibits cluster formation, identifying PLK-1 as a regulator of this process. Phosphorylation by PLK-1 on PAR-3 must be relieved to allow cluster formation and polarity establishment. We hypothesized that a phosphatase counteracts PLK-1 phosphorylation during polarity establishment. To test this hypothesis we treated the embryos with phosphatases inhibitors and found that inhibits polarity establishment. To identify the phosphatases, we are currently performing RNAi interference and biochemical approaches. Our data will help to better understand how PLK-1 regulates cell polarity establishment.

Kress E et al. (2013) Cell Cycle "When the Swiss clock goes wrong: regulation of Aurora A by UBXN-2/CDC-48."

Gotta M, Kress E

[Cell Cycle, 2013]

Barbieri S et al. (2023) Trends Cell Biol "Order from chaos: cellular asymmetries explained with modelling."

Barbieri S, Gotta M

[Trends Cell Biol, 2023]

Molecules inside cells are subject to physical forces and undergo biochemical interactions, continuously changing their physical properties and dynamics. Despite this, cells achieve highly ordered molecular patterns that are crucial to regulate various cellular functions and to specify cell fate. In the Caenorhabditis elegans one-cell embryo, protein asymmetries are established in the narrow time window of a cell division. What are the mechanisms that allow molecules to establish asymmetries, defying the randomness imposed by Brownian motion? Mathematical and computational models have paved the way to the understanding of protein dynamics up to the 'single-molecule level' when resolution represents an issue for precise experimental measurements. Here we review the models that interpret cortical and cytoplasmic asymmetries in the one-cell C. elegans embryo.

Kress E et al. (2011) Nat Cell Biol "Aurora A in cell division: kinase activity not required."

Kress E, Gotta M

[Nat Cell Biol, 2011]

Aurora A kinase is a key regulator of cell division, whose functions were attributed to its ability to phosphorylate diverse substrates. Aurora A is now shown to have a kinase-independent role in the regulation of chromatin-mediated microtubule assembly.

Noatynska A et al. (2012) Essays Biochem "Cell polarity and asymmetric cell division: the C. elegans early embryo."

Gotta M, Noatynska A

[Essays Biochem, 2012]

Cell polarity is crucial for many functions including cell migration, tissue organization and asymmetric cell division. In animal cells, cell polarity is controlled by the highly conserved PAR (PARtitioning defective) proteins. par genes have been identified in Caenorhabditis elegans in screens for maternal lethal mutations that disrupt cytoplasmic partitioning and asymmetric division. Although PAR proteins were identified more than 20 years ago, our understanding on how they regulate polarity and how they are regulated is still incomplete. In this chapter we review our knowledge of the processes of cell polarity establishment and maintenance, and asymmetric cell division in the early C. elegans embryo. We discuss recent findings that highlight new players in cell polarity and/or reveal the molecular details on how PAR proteins regulate polarity processes.