Developmental Cell
Previews Protein Clustering Shapes Polarity Protein Gradients Edwin Munro1,* 1Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2017.08.006
In this issue of Developmental Cell, Dickinson et al. (2017) and Rodriguez et al. (2017), along with Wang et al. (2017) in Nature Cell Biology, show how PAR protein oligomerization can dynamically couple protein diffusion and transport by cortical flow to control kinase activity gradients and polarity in the C. elegans zygote. Dynamic clustering of cell surface proteins is emerging as a key mode of physiological control in animal cells. Two papers in this issue of Developmental Cell (Dickinson et al., 2017 and Rodriguez et al., 2017), and a third in Nature Cell Biology (Wang et al., 2017), highlight a central role for the oligomerization of PAR proteins in forming and shaping gradients of kinase activity in the polarizing C. elegans zygote. The PAR polarity proteins are a highly conserved network of scaffolding proteins, adaptors, and enzymes that localize asymmetrically in polarized cells and control various downstream targets to polarize cell and tissue behaviors (Goldstein and Macara, 2007). At the heart of the PAR network is the apical or anterior PAR complex, which consists of the oligomeric scaffold PAR-3, the small GTPase CDC-42, the adaptor PAR-6, and the kinase aPKC (PKC-3 in C. elegans). The business end of this complex is PKC-3, which phosphorylates many different target proteins to control their localizations and/or activities. PKC-3 forms a heterodimer with PAR-6, which can bind either to PAR-3 or to CDC-42. A key challenge in the field has been to understand how a complex set of interactions among these four proteins shapes the localization and activity of PKC-3. The three studies by Dickinson, Rodriguez, Wang and colleagues shed new light on how this works in the polarizing C. elegans zygote (Figure 1). In the C. elegans zygote, PAR asymmetries are established during interphase in response to a transient sperm-derived cue and they are then maintained by cross-inhibition between anterior and posterior PAR proteins. Early studies showed that the initial segregation of anterior PARs involves active transport
by actomyosin-dependent cortical flows (Munro et al., 2004). Subsequent biophysical studies revealed that PAR-6/PKC-3 exchange dynamically between the cytoplasm and the cell surface and that much (but not all) of PAR-6/PKC-3 exhibits rapid diffusion characteristic of membrane-associated proteins (Goehring et al., 2011; Robin et al., 2014). Quantitative models suggest that cortical flows could be sufficient to segregate PAR-6/ PKC-3 if individual molecules of PAR-6/ PKC-3 were tightly coupled to the moving flow. But this raises a fundamental question: How can a protein both diffuse rapidly within the plasma membrane and couple tightly to the underlying actomyosin cortex, unless the entire plasma membrane was moving along with the cortical flow? Indeed, classical studies showed that bulk flow of membrane does not accompany cortical flow in other contexts, such as the leading edge of motile cells (Kucik et al., 1990). An important hint at the answer to this question came from earlier observations that PAR-6/PKC-3 accumulates in two forms: clustered (i.e., punctate) and diffuse (Beers and Kemphues, 2006). Extending these earlier studies to live cells, Wang et al. (2017) showed that clusters of PAR-6/PKC-3 partially colocalize with clusters of PAR-3, while the diffuse pool of PAR-6/PKC-3 appears to colocalize with CDC-42 in regions enriched for PtdIns(4,5)P2. The diffuse pool requires CDC-42 for localization, whereas both pools require PAR-3 (Beers and Kemphues, 2006; Wang et al., 2017). This suggested that the two pools represent PAR-6/PKC-3 heterodimers that are differently complexed with PAR-3 and CDC-42, but it remained unclear what the relationship is between these com-
plexes, which of them contains the active form of PKC-3, and how their distributions are shaped by cortical flow. Using an elegant single-cell biochemical approach (scSimPull) to capture and visualize complexes released by lysis of single, precisely staged live cells, Dickinson et al. (2017) were able to detect variably sized oligomers of both PAR-3 and PAR-6/PKC-3, whose mean sizes increase during polarity establishment and decrease during polarity maintenance, as observed in vivo. Importantly, they could detect robust association between PAR-6/PKC-3 and PAR-3 oligomers, as well as a sharp increase in the fractional binding of PAR-6/PKC-3 to PAR-3 oligomers with oligomer size, suggesting that PAR-3 oligomers act as a scaffold to promote cooperative recruitment/oligomerization of PAR-6/PKC-3. In complementary experiments, Rodriguez et al. (2017) used genetic and pharmacological methods to tease apart the contributions of PAR-3 and CDC-42 to PKC-3 recruitment and activity. They found that inhibiting PKC-3 activity blocks the accumulation of clustered, but not diffuse, PAR-6/PKC-3. Indeed, while the accumulation of inactive PKC-3 still depends on CDC-42, it no longer requires PAR-3. On the other hand, by forcing PKC-3 onto the membrane and using displacement of the posterior PAR protein PAR-2 as a sensitive readout for local PKC-3 activity, Rodriguez et al. (2017) showed that PKC-3 activity requires both CDC-42 and PAR-6, but not PAR-3. Indeed, PKC-3 activity is enhanced in the absence of PAR-3, consistent with the finding that PAR-3 can bind and inhibit PKC-3 in other contexts (for example, see Soriano et al., 2016). Altogether, these results suggest that PAR-3 recruits active cytoplasmic
Developmental Cell 42, August 21, 2017 ª 2017 Published by Elsevier Inc. 309
Developmental Cell
Previews
Figure 1. Dynamic Segregation of Clustered PAR-3 Shapes a Gradient of PKC-3 Kinase Activity (A) Hypothetical kinetic pathway for recruitment of an active diffusible form of PKC-3 by PAR-3 oligomers: (1) PAR-3 oligomers promote recruitment/assembly of PAR-6/PKC-3 clusters; (2) PAR-6/ PKC-3 heterodimers are transferred into an active and rapidly diffusing complex with active CDC-42; (3) PAR-6/PKC-3 dimers diffuse away from the PAR-3 ‘‘source’’ and dissociate to form a gradient of active kinase. (B) During polarity establishment, active transport by cortical flow and growth of PAR-3 oligomers concentrate PAR-3 and associated clusters of PAR-6/PKC-3 at the anterior pole to form the source for a spatial gradient of diffusible PAR-6/PKC-3. During polarity maintenance, shrinkage of PAR-3 clusters and increased CDC-42 activity promote a shift toward active diffusible PAR-6/PKC-3.
PAR-6/PKC-3 dimers into an inactive cortical complex. CDC-42 cannot directly recruit active PKC-3, but somehow PAR-3 can promote the recruitment of PKC-3 into an active complex with CDC42. This dovetails nicely with the results of recent single-molecule imaging experiments, which suggest that a small amount of PAR-3 is both necessary and sufficient for rapid local recruitment of PAR-6/ PKC-3 into a complex with CDC-42 (Sailer et al., 2015). Why active CDC-42 cannot bind to active PAR-6/PKC-3 dimers, and how small amounts of PAR-3 can efficiently promote the local association of PAR-6/PKC-3 with CDC-42, remains an interesting puzzle. Regardless, the implication is that the spatial distribution of PAR-3 defines the source domain for local formation of an active CDC-42/PAR-6/ 310 Developmental Cell 42, August 21, 2017
PKC-3 complex, which diffuses away and dissociates to form a gradient of active PKC-3. Why is clustering of Par-3 particularly important? Rodriguez et al. (2017) show that a membrane-tethered monomeric form of PAR-3 is sufficient to promote local recruitment of diffuse PAR-6/ PKC-3. However, neither the monomeric form of PAR-3 nor the diffuse pool of PAR-6/PKC-3 is segregated by cortical flows during polarity establishment. In other experiments, Rodriguez et al. show that reducing the diffuse pool of PAR-6/ PKC-3 (by depleting CDC-42) enhances segregation of PAR-6/PKC-3 by cortical flow, whereas increasing the diffuse pool (by inhibiting PKC-3) reduces segregation. These results suggest that clustering of PAR-3 is essential for its segregation by
cortical flow, whereas binding of PAR-6/ PKC-3 to clustered PAR-3 promotes the efficient segregation of PAR-6/ PKC-3 to the anterior pole during polarity establishment. By combining fluorescence recovery after photobleaching with particle image velocimetry, Wang et al. (2017) find that the effective residence times of PAR-3 and local correlations of PAR-3 cluster movement with cortical flow both increase with the mean sizes of PAR-3 clusters. Dickinson et al. (2017) use particletracking analysis to show even more directly that the cortical residence times of individual PAR-3 clusters, and their persistence of motion in cortical flow (the ratio of directed to uncorrelated motion), increase with cluster size. Thus, clustering of PAR-3 increases both cluster residence time (likely through simple avidity effects) and local coupling to actomyosin, to promote efficient transport by cortical flow. Finally, work from all three groups provides insights into the mechanisms that control the size of PAR-3 clusters and the partitioning of PAR-6/PKC-3 into clustered and diffuse assemblies as the zygote progresses through polarity establishment and maintenance phases. Dickinson et al. (2017) identify the Polo Kinase PLK-1 as a key regulator of PAR-3 oligomerization. PLK-1 phosphorylates PAR-3 at two sites in its N terminus, and this phosphorylation is both required and sufficient to drive shrinkage of PAR-3 clusters during mitosis/maintenance phase (when PLK-1 is known to be active in mammalian cells). Further, CDC-42 activity is known to increase at the anterior pole during maintenance phase, and both Wang et al. (2017) and Rodriguez et al. (2017) show that increasing CDC-42 activity drives a shift toward diffuse PAR-6/ PKC-3, whereas decreasing CDC-42 activity drives a shift toward clustered PAR-6/PKC-3, consistent with a scenario in which active CDC-42 can recruit PAR-6/PKC-3 away from PAR-3 clusters into an active complex. Thus, the zygote modulates PLK-1 and CDC-42 activities to favor highly clustered PAR-3 and PAR-6/PKC-3 and cortical transport during polarity establishment and to promote a shift toward active and diffusive PKC-3 during polarity maintenance. Together, these results provide an elegant answer to the challenge of how
Developmental Cell
Previews to couple rapidly diffusing proteins to cortical flow: tight coupling of PAR-3 oligomers to the actomyosin cortex provides for rapid initial segregation of both PAR-3 and associated clusters of PAR-6/PKC-3 (Figure 1). Once segregated, PAR-3 defines a localized source for a rapidly diffusing pool of active PKC-3 that can efficiently act on targets distributed throughout a broader domain. It will be interesting to see whether variants of this mechanism operate in the many other contexts in which gradients of aPKC control the essential behaviors of polarized cells.
Robin, F.B., McFadden, W.M., Yao, B., and Munro, E.M. (2014). Nat. Methods 11, 677–682.
REFERENCES Beers, M., and Kemphues, K. (2006). Development 133, 3745–3754. Dickinson, D.J., Schwager, F., Pintard, L., Gotta, M., and Goldstein, B. (2017). Dev. Cell 42, this issue, 416–434. Goehring, N.W., Hoege, C., Grill, S.W., and Hyman, A.A. (2011). J. Cell Biol. 193, 583–594. Goldstein, B., and Macara, I.G. (2007). Dev. Cell 13, 609–622. Kucik, D.F., Elson, E.L., and Sheetz, M.P. (1990). J. Cell Biol. 111, 1617–1622. Munro, E., Nance, J., and Priess, J.R. (2004). Dev. Cell 7, 413–424.
Rodriguez, J., Peglion, F., Martin, J., Hubatsche, L., Reich, J., Hirani, N., Gubieda, A.G., Roffey, J., Fernandez, A.R., St Johnston, D., et al. (2017). Dev. Cell 42, this issue, 400–415. Sailer, A., Anneken, A., Li, Y., Lee, S., and Munro, E. (2015). Dev. Cell 35, 131–142. Soriano, E.V., Ivanova, M.E., Fletcher, G., Riou, P., Knowles, P.P., Barnouin, K., Purkiss, A., Kostelecky, B., Saiu, P., Linch, M., et al. (2016). Dev. Cell 38, 384–398. Wang, S.-C., Low, T.Y.F., Nishimura, Y., Gole, L., Yu, W., and Motegi, F. (2017). Nat. Cell Biol. 19, 988–995.
Patchy Growth Control Hugo Stocker1,*
€rich, Zu € rich, Switzerland of Molecular Systems Biology, ETH Zu *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2017.08.007
1Institute
TORC1 is arguably the best-studied regulator of cellular growth in eukaryotes. However, its activity has not been assessed in vivo in developing multicellular organisms. Two studies in this issue of Developmental Cell (Kim et al., 2017; Romero-Pozuelo et al., 2017) surprisingly reveal a patchy, cell-cycle-controlled pattern of TORC1 activation in Drosophila imaginal discs. Target of rapamycin complex 1 (TORC1) controls growth of eukaryotic cells by promoting anabolic (e.g., protein and lipid biosynthesis) and inhibiting catabolic (e.g., autophagy) processes (Wolfson and Sabatini, 2017). An intricate two-step mechanism ensures that TORC1 (and thus biomass accumulation) is only activated when a cell has sufficient resources (amino acids) and receives growth-permissive signals. Given this, one might expect that TORC1 is constantly and homogeneously active in a growing organ. However, monitoring TORC1 activity in situ has not been possible in multicellular organisms so far. The standard assay for the TORC1 activation status consists in determining the phosphorylation of the ribosomal protein S6 kinase (S6K), a direct target of TORC1’s kinase activity. The proportion of phosphorylated S6K—typically determined by western blotting—is a good measure for the average TORC1
activity in the analyzed tissue but fails to provide positional information. In two papers in this issue of Developmental Cell, research teams have used an indirect approach to detect TORC1 activity in the model organism Drosophila melanogaster, reasoning that the phosphorylation of an S6K target protein should also reflect TORC1 activity. They generated antisera against phosphorylated peptides of the ribosomal protein S6 (RpS6), which—in contrast to the anti-phospho-S6K antibody—work in situ. A series of control experiments indicated that the anti-phospho-RpS6 antibodies faithfully detect TORC1 activity in imaginal discs during development. Surprisingly, the observed TORC1 activation patterns are anything but uniform. Whereas RpS6 is phosphorylated in groups of cells posterior to the morphogenetic furrow in eye imaginal discs (Kim et al., 2017), the antibody staining produced a patchy pattern in
wing imaginal discs (Romero-Pozuelo et al., 2017). Phospho-RpS6-positive cells in the eye imaginal discs correspond to S phase cells in the second mitotic wave (SMW, a synchronized round of cell cycle in all undetermined cells just posterior to the morphogenetic furrow). The two main upstream branches of TORC1 regulation— amino acid sensing via the RagA/RagC complex and insulin signaling via TSC/ Rheb—are required for TORC1 activation in the SMW. However, Kim et al. (2017) find that neither insulin nor amino acid signaling is sufficient to induce TORC1 activity in eye imaginal discs. Hedgehog (Hh) signaling has been known to control cell-cycle progression in the SMW and is believed to do so indirectly via the regulation of Notch signaling (Baonza and Freeman, 2005). Surprisingly, the authors find that regulation of TORC1 by Hh signaling appears to be independent of Notch (Kim et al., 2017).
Developmental Cell 42, August 21, 2017 ª 2017 Elsevier Inc. 311
Previews Protein Clustering Shapes Polarity Protein Gradients Edwin Munro1,* 1Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2017.08.006
In this issue of Developmental Cell, Dickinson et al. (2017) and Rodriguez et al. (2017), along with Wang et al. (2017) in Nature Cell Biology, show how PAR protein oligomerization can dynamically couple protein diffusion and transport by cortical flow to control kinase activity gradients and polarity in the C. elegans zygote. Dynamic clustering of cell surface proteins is emerging as a key mode of physiological control in animal cells. Two papers in this issue of Developmental Cell (Dickinson et al., 2017 and Rodriguez et al., 2017), and a third in Nature Cell Biology (Wang et al., 2017), highlight a central role for the oligomerization of PAR proteins in forming and shaping gradients of kinase activity in the polarizing C. elegans zygote. The PAR polarity proteins are a highly conserved network of scaffolding proteins, adaptors, and enzymes that localize asymmetrically in polarized cells and control various downstream targets to polarize cell and tissue behaviors (Goldstein and Macara, 2007). At the heart of the PAR network is the apical or anterior PAR complex, which consists of the oligomeric scaffold PAR-3, the small GTPase CDC-42, the adaptor PAR-6, and the kinase aPKC (PKC-3 in C. elegans). The business end of this complex is PKC-3, which phosphorylates many different target proteins to control their localizations and/or activities. PKC-3 forms a heterodimer with PAR-6, which can bind either to PAR-3 or to CDC-42. A key challenge in the field has been to understand how a complex set of interactions among these four proteins shapes the localization and activity of PKC-3. The three studies by Dickinson, Rodriguez, Wang and colleagues shed new light on how this works in the polarizing C. elegans zygote (Figure 1). In the C. elegans zygote, PAR asymmetries are established during interphase in response to a transient sperm-derived cue and they are then maintained by cross-inhibition between anterior and posterior PAR proteins. Early studies showed that the initial segregation of anterior PARs involves active transport
by actomyosin-dependent cortical flows (Munro et al., 2004). Subsequent biophysical studies revealed that PAR-6/PKC-3 exchange dynamically between the cytoplasm and the cell surface and that much (but not all) of PAR-6/PKC-3 exhibits rapid diffusion characteristic of membrane-associated proteins (Goehring et al., 2011; Robin et al., 2014). Quantitative models suggest that cortical flows could be sufficient to segregate PAR-6/ PKC-3 if individual molecules of PAR-6/ PKC-3 were tightly coupled to the moving flow. But this raises a fundamental question: How can a protein both diffuse rapidly within the plasma membrane and couple tightly to the underlying actomyosin cortex, unless the entire plasma membrane was moving along with the cortical flow? Indeed, classical studies showed that bulk flow of membrane does not accompany cortical flow in other contexts, such as the leading edge of motile cells (Kucik et al., 1990). An important hint at the answer to this question came from earlier observations that PAR-6/PKC-3 accumulates in two forms: clustered (i.e., punctate) and diffuse (Beers and Kemphues, 2006). Extending these earlier studies to live cells, Wang et al. (2017) showed that clusters of PAR-6/PKC-3 partially colocalize with clusters of PAR-3, while the diffuse pool of PAR-6/PKC-3 appears to colocalize with CDC-42 in regions enriched for PtdIns(4,5)P2. The diffuse pool requires CDC-42 for localization, whereas both pools require PAR-3 (Beers and Kemphues, 2006; Wang et al., 2017). This suggested that the two pools represent PAR-6/PKC-3 heterodimers that are differently complexed with PAR-3 and CDC-42, but it remained unclear what the relationship is between these com-
plexes, which of them contains the active form of PKC-3, and how their distributions are shaped by cortical flow. Using an elegant single-cell biochemical approach (scSimPull) to capture and visualize complexes released by lysis of single, precisely staged live cells, Dickinson et al. (2017) were able to detect variably sized oligomers of both PAR-3 and PAR-6/PKC-3, whose mean sizes increase during polarity establishment and decrease during polarity maintenance, as observed in vivo. Importantly, they could detect robust association between PAR-6/PKC-3 and PAR-3 oligomers, as well as a sharp increase in the fractional binding of PAR-6/PKC-3 to PAR-3 oligomers with oligomer size, suggesting that PAR-3 oligomers act as a scaffold to promote cooperative recruitment/oligomerization of PAR-6/PKC-3. In complementary experiments, Rodriguez et al. (2017) used genetic and pharmacological methods to tease apart the contributions of PAR-3 and CDC-42 to PKC-3 recruitment and activity. They found that inhibiting PKC-3 activity blocks the accumulation of clustered, but not diffuse, PAR-6/PKC-3. Indeed, while the accumulation of inactive PKC-3 still depends on CDC-42, it no longer requires PAR-3. On the other hand, by forcing PKC-3 onto the membrane and using displacement of the posterior PAR protein PAR-2 as a sensitive readout for local PKC-3 activity, Rodriguez et al. (2017) showed that PKC-3 activity requires both CDC-42 and PAR-6, but not PAR-3. Indeed, PKC-3 activity is enhanced in the absence of PAR-3, consistent with the finding that PAR-3 can bind and inhibit PKC-3 in other contexts (for example, see Soriano et al., 2016). Altogether, these results suggest that PAR-3 recruits active cytoplasmic
Developmental Cell 42, August 21, 2017 ª 2017 Published by Elsevier Inc. 309
Developmental Cell
Previews
Figure 1. Dynamic Segregation of Clustered PAR-3 Shapes a Gradient of PKC-3 Kinase Activity (A) Hypothetical kinetic pathway for recruitment of an active diffusible form of PKC-3 by PAR-3 oligomers: (1) PAR-3 oligomers promote recruitment/assembly of PAR-6/PKC-3 clusters; (2) PAR-6/ PKC-3 heterodimers are transferred into an active and rapidly diffusing complex with active CDC-42; (3) PAR-6/PKC-3 dimers diffuse away from the PAR-3 ‘‘source’’ and dissociate to form a gradient of active kinase. (B) During polarity establishment, active transport by cortical flow and growth of PAR-3 oligomers concentrate PAR-3 and associated clusters of PAR-6/PKC-3 at the anterior pole to form the source for a spatial gradient of diffusible PAR-6/PKC-3. During polarity maintenance, shrinkage of PAR-3 clusters and increased CDC-42 activity promote a shift toward active diffusible PAR-6/PKC-3.
PAR-6/PKC-3 dimers into an inactive cortical complex. CDC-42 cannot directly recruit active PKC-3, but somehow PAR-3 can promote the recruitment of PKC-3 into an active complex with CDC42. This dovetails nicely with the results of recent single-molecule imaging experiments, which suggest that a small amount of PAR-3 is both necessary and sufficient for rapid local recruitment of PAR-6/ PKC-3 into a complex with CDC-42 (Sailer et al., 2015). Why active CDC-42 cannot bind to active PAR-6/PKC-3 dimers, and how small amounts of PAR-3 can efficiently promote the local association of PAR-6/PKC-3 with CDC-42, remains an interesting puzzle. Regardless, the implication is that the spatial distribution of PAR-3 defines the source domain for local formation of an active CDC-42/PAR-6/ 310 Developmental Cell 42, August 21, 2017
PKC-3 complex, which diffuses away and dissociates to form a gradient of active PKC-3. Why is clustering of Par-3 particularly important? Rodriguez et al. (2017) show that a membrane-tethered monomeric form of PAR-3 is sufficient to promote local recruitment of diffuse PAR-6/ PKC-3. However, neither the monomeric form of PAR-3 nor the diffuse pool of PAR-6/PKC-3 is segregated by cortical flows during polarity establishment. In other experiments, Rodriguez et al. show that reducing the diffuse pool of PAR-6/ PKC-3 (by depleting CDC-42) enhances segregation of PAR-6/PKC-3 by cortical flow, whereas increasing the diffuse pool (by inhibiting PKC-3) reduces segregation. These results suggest that clustering of PAR-3 is essential for its segregation by
cortical flow, whereas binding of PAR-6/ PKC-3 to clustered PAR-3 promotes the efficient segregation of PAR-6/ PKC-3 to the anterior pole during polarity establishment. By combining fluorescence recovery after photobleaching with particle image velocimetry, Wang et al. (2017) find that the effective residence times of PAR-3 and local correlations of PAR-3 cluster movement with cortical flow both increase with the mean sizes of PAR-3 clusters. Dickinson et al. (2017) use particletracking analysis to show even more directly that the cortical residence times of individual PAR-3 clusters, and their persistence of motion in cortical flow (the ratio of directed to uncorrelated motion), increase with cluster size. Thus, clustering of PAR-3 increases both cluster residence time (likely through simple avidity effects) and local coupling to actomyosin, to promote efficient transport by cortical flow. Finally, work from all three groups provides insights into the mechanisms that control the size of PAR-3 clusters and the partitioning of PAR-6/PKC-3 into clustered and diffuse assemblies as the zygote progresses through polarity establishment and maintenance phases. Dickinson et al. (2017) identify the Polo Kinase PLK-1 as a key regulator of PAR-3 oligomerization. PLK-1 phosphorylates PAR-3 at two sites in its N terminus, and this phosphorylation is both required and sufficient to drive shrinkage of PAR-3 clusters during mitosis/maintenance phase (when PLK-1 is known to be active in mammalian cells). Further, CDC-42 activity is known to increase at the anterior pole during maintenance phase, and both Wang et al. (2017) and Rodriguez et al. (2017) show that increasing CDC-42 activity drives a shift toward diffuse PAR-6/ PKC-3, whereas decreasing CDC-42 activity drives a shift toward clustered PAR-6/PKC-3, consistent with a scenario in which active CDC-42 can recruit PAR-6/PKC-3 away from PAR-3 clusters into an active complex. Thus, the zygote modulates PLK-1 and CDC-42 activities to favor highly clustered PAR-3 and PAR-6/PKC-3 and cortical transport during polarity establishment and to promote a shift toward active and diffusive PKC-3 during polarity maintenance. Together, these results provide an elegant answer to the challenge of how
Developmental Cell
Previews to couple rapidly diffusing proteins to cortical flow: tight coupling of PAR-3 oligomers to the actomyosin cortex provides for rapid initial segregation of both PAR-3 and associated clusters of PAR-6/PKC-3 (Figure 1). Once segregated, PAR-3 defines a localized source for a rapidly diffusing pool of active PKC-3 that can efficiently act on targets distributed throughout a broader domain. It will be interesting to see whether variants of this mechanism operate in the many other contexts in which gradients of aPKC control the essential behaviors of polarized cells.
Robin, F.B., McFadden, W.M., Yao, B., and Munro, E.M. (2014). Nat. Methods 11, 677–682.
REFERENCES Beers, M., and Kemphues, K. (2006). Development 133, 3745–3754. Dickinson, D.J., Schwager, F., Pintard, L., Gotta, M., and Goldstein, B. (2017). Dev. Cell 42, this issue, 416–434. Goehring, N.W., Hoege, C., Grill, S.W., and Hyman, A.A. (2011). J. Cell Biol. 193, 583–594. Goldstein, B., and Macara, I.G. (2007). Dev. Cell 13, 609–622. Kucik, D.F., Elson, E.L., and Sheetz, M.P. (1990). J. Cell Biol. 111, 1617–1622. Munro, E., Nance, J., and Priess, J.R. (2004). Dev. Cell 7, 413–424.
Rodriguez, J., Peglion, F., Martin, J., Hubatsche, L., Reich, J., Hirani, N., Gubieda, A.G., Roffey, J., Fernandez, A.R., St Johnston, D., et al. (2017). Dev. Cell 42, this issue, 400–415. Sailer, A., Anneken, A., Li, Y., Lee, S., and Munro, E. (2015). Dev. Cell 35, 131–142. Soriano, E.V., Ivanova, M.E., Fletcher, G., Riou, P., Knowles, P.P., Barnouin, K., Purkiss, A., Kostelecky, B., Saiu, P., Linch, M., et al. (2016). Dev. Cell 38, 384–398. Wang, S.-C., Low, T.Y.F., Nishimura, Y., Gole, L., Yu, W., and Motegi, F. (2017). Nat. Cell Biol. 19, 988–995.
Patchy Growth Control Hugo Stocker1,*
€rich, Zu € rich, Switzerland of Molecular Systems Biology, ETH Zu *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2017.08.007
1Institute
TORC1 is arguably the best-studied regulator of cellular growth in eukaryotes. However, its activity has not been assessed in vivo in developing multicellular organisms. Two studies in this issue of Developmental Cell (Kim et al., 2017; Romero-Pozuelo et al., 2017) surprisingly reveal a patchy, cell-cycle-controlled pattern of TORC1 activation in Drosophila imaginal discs. Target of rapamycin complex 1 (TORC1) controls growth of eukaryotic cells by promoting anabolic (e.g., protein and lipid biosynthesis) and inhibiting catabolic (e.g., autophagy) processes (Wolfson and Sabatini, 2017). An intricate two-step mechanism ensures that TORC1 (and thus biomass accumulation) is only activated when a cell has sufficient resources (amino acids) and receives growth-permissive signals. Given this, one might expect that TORC1 is constantly and homogeneously active in a growing organ. However, monitoring TORC1 activity in situ has not been possible in multicellular organisms so far. The standard assay for the TORC1 activation status consists in determining the phosphorylation of the ribosomal protein S6 kinase (S6K), a direct target of TORC1’s kinase activity. The proportion of phosphorylated S6K—typically determined by western blotting—is a good measure for the average TORC1
activity in the analyzed tissue but fails to provide positional information. In two papers in this issue of Developmental Cell, research teams have used an indirect approach to detect TORC1 activity in the model organism Drosophila melanogaster, reasoning that the phosphorylation of an S6K target protein should also reflect TORC1 activity. They generated antisera against phosphorylated peptides of the ribosomal protein S6 (RpS6), which—in contrast to the anti-phospho-S6K antibody—work in situ. A series of control experiments indicated that the anti-phospho-RpS6 antibodies faithfully detect TORC1 activity in imaginal discs during development. Surprisingly, the observed TORC1 activation patterns are anything but uniform. Whereas RpS6 is phosphorylated in groups of cells posterior to the morphogenetic furrow in eye imaginal discs (Kim et al., 2017), the antibody staining produced a patchy pattern in
wing imaginal discs (Romero-Pozuelo et al., 2017). Phospho-RpS6-positive cells in the eye imaginal discs correspond to S phase cells in the second mitotic wave (SMW, a synchronized round of cell cycle in all undetermined cells just posterior to the morphogenetic furrow). The two main upstream branches of TORC1 regulation— amino acid sensing via the RagA/RagC complex and insulin signaling via TSC/ Rheb—are required for TORC1 activation in the SMW. However, Kim et al. (2017) find that neither insulin nor amino acid signaling is sufficient to induce TORC1 activity in eye imaginal discs. Hedgehog (Hh) signaling has been known to control cell-cycle progression in the SMW and is believed to do so indirectly via the regulation of Notch signaling (Baonza and Freeman, 2005). Surprisingly, the authors find that regulation of TORC1 by Hh signaling appears to be independent of Notch (Kim et al., 2017).
Developmental Cell 42, August 21, 2017 ª 2017 Elsevier Inc. 311
