The Drosophila neuroblast polarity cycle at a glance.

Drosophila neural stem cells, or neuroblasts, rapidly proliferate during embryonic and larval development to populate the central nervous system. Neuroblasts divide asymmetrically to create cellular diversity, with each division producing one sibling cell that retains the neuroblast fate and another that differentiates into glia or neurons. This asymmetric outcome is mediated by the transient polarization of numerous factors to the cell cortex during mitosis. The powerful genetics and outstanding imaging tractability of the neuroblast make it an excellent model system for studying the mechanisms of cell polarity. This Cell Science at a Glance article and the accompanying poster explore the phases of the neuroblast polarity cycle and the regulatory circuits that control them. We discuss the key features of the cycle - the targeted recruitment of proteins to specific regions of the plasma membrane and multiple phases of highly dynamic actomyosin-dependent cortical flows that pattern both protein distribution and membrane structure.

Cooperative regulation of C1-domain membrane recruitment polarizes atypical protein kinase C.

Recruitment of the Par complex protein atypical protein kinase C (aPKC) to a specific membrane domain is a key step in the polarization of animal cells. While numerous proteins and phospholipids interact with aPKC, how these interactions cooperate to control its membrane recruitment has been unknown. Here, we identify aPKC's C1 domain as a phospholipid interaction module that targets aPKC to the membrane of Drosophila neural stem cells (NSCs). The isolated C1 binds the NSC membrane in an unpolarized manner during interphase and mitosis and is uniquely sufficient among aPKC domains for targeting. Other domains, including the catalytic module and those that bind the upstream regulators Par-6 and Bazooka, restrict C1's membrane targeting activity-spatially and temporally-to the apical NSC membrane during mitosis. Our results suggest that aPKC polarity results from cooperative activation of autoinhibited C1-mediated membrane binding activity.

Consumption of a polarized membrane reservoir drives asymmetric membrane expansion during the unequal divisions of neural stem cells.

The asymmetric divisions of Drosophila neural stem cells (NSCs) produce unequally sized siblings, with most volume directed into the sibling that retains the NSC fate. Sibling size asymmetry results from the preferential expansion of the NSC sibling surface during division. Here, we show that a polarized membrane reservoir constructed by the NSC in early mitosis provides the source for expansion. The reservoir is formed from membrane domains that contain folds and microvilli that become polarized by apically directed cortical flows of actomyosin early in mitosis. When furrow ingression begins and internal pressure increases, the stores of membrane within the apical reservoir are rapidly consumed. Expansion is substantially diminished in NSCs that lack a reservoir, and membrane expansion equalizes when the reservoir is not polarized. Our results suggest that the cortical flows that remodel the plasma membrane during asymmetric cell division function to satisfy the dynamic surface area requirements of unequally dividing cells.

Negative cooperativity underlies dynamic assembly of the Par complex regulators Cdc42 and Par-3.

The Par complex polarizes diverse animal cells through the concerted action of multiple regulators. Binding to the multi-PDZ domain containing protein Par-3 couples the complex to cortical flows that construct the Par membrane domain. Once localized properly, the complex is thought to transition from Par-3 to the Rho GTPase Cdc42 to activate the complex. While this transition is a critical step in Par-mediated polarity, little is known about how it occurs. Here, we used a biochemical reconstitution approach with purified, intact Par complex and qualitative binding assays and found that Par-3 and Cdc42 exhibit strong negative cooperativity for the Par complex. The energetic coupling arises from interactions between the second and third PDZ protein interaction domains of Par-3 and the aPKC Kinase-PBM (PDZ binding motif) that mediate the displacement of Cdc42 from the Par complex. Our results indicate that Par-3, Cdc42, Par-6, and aPKC are the minimal components that are sufficient for this transition to occur and that no external factors are required. Our findings provide the mechanistic framework for understanding a critical step in the regulation of Par complex polarization and activity.

Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex.

The animal cell polarity regulator Par-3 recruits the Par complex (consisting of Par-6 and atypical PKC, aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it is unclear how each of these interactions contributes to the overall binding. Using a purified, intact Par complex and a quantitative binding assay, here, we found that the energy required for this interaction is provided by the second and third PDZ protein interaction domains of Par-3. We show that both Par-3 PDZ domains bind to the PDZ-binding motif of aPKC in the Par complex, with additional binding energy contributed from the adjacent catalytic domain of aPKC. In addition to highlighting the role of Par-3 PDZ domain interactions with the aPKC kinase domain and PDZ-binding motif in stabilizing Par-3-Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization. These results provide new insights into the energetic determinants and structural stoichiometry of the Par-3-Par complex assembly.

Phases of cortical actomyosin dynamics coupled to the neuroblast polarity cycle.

The Par complex dynamically polarizes to the apical cortex of asymmetrically dividing Drosophila neuroblasts where it directs fate determinant segregation. Previously, we showed that apically directed cortical movements that polarize the Par complex require F-actin (Oon and Prehoda, 2019). Here, we report the discovery of cortical actomyosin dynamics that begin in interphase when the Par complex is cytoplasmic but ultimately become tightly coupled to cortical Par dynamics. Interphase cortical actomyosin dynamics are unoriented and pulsatile but rapidly become sustained and apically-directed in early mitosis when the Par protein aPKC accumulates on the cortex. Apical actomyosin flows drive the coalescence of aPKC into an apical cap that depolarizes in anaphase when the flow reverses direction. Together with the previously characterized role of anaphase flows in specifying daughter cell size asymmetry, our results indicate that multiple phases of cortical actomyosin dynamics regulate asymmetric cell division.

Actin-dependent membrane polarization reveals the mechanical nature of the neuroblast polarity cycle.

The Par complex directs fate-determinant segregation from the apical membrane of asymmetrically dividing Drosophila neuroblasts. While the physical interactions that recruit the Par complex have been extensively studied, little is known about how the membrane itself behaves during polarization. We examined the membrane dynamics of neuroblasts and surrounding cells using a combination of super-resolution and time-lapse imaging, revealing cellular-scale movements of diverse membrane features during asymmetric division cycles. Membrane domains that are distributed across the neuroblast membrane in interphase become polarized in early mitosis, where they mediate formation of cortical patches of the Par protein atypical protein kinase C (aPKC). Membrane and protein polarity cycles are precisely synchronized and are generated by extensive actin-dependent forces that deform the surrounding tissue. In addition to suggesting a role for the membrane in asymmetric division, our results reveal the mechanical nature of the neuroblast polarity cycle.

Evolution of fold switching in a metamorphic protein.

Metamorphic proteins switch between different folds, defying the protein folding paradigm. It is unclear how fold switching arises during evolution. With ancestral reconstruction and nuclear magnetic resonance, we studied the evolution of the metamorphic human protein XCL1, which has two distinct folds with different functions, making it an unusual member of the chemokine family, whose members generally adopt one conserved fold. XCL1 evolved from an ancestor with the chemokine fold. Evolution of a dimer interface, changes in structural constraints and molecular strain, and alteration of intramolecular protein contacts drove the evolution of metamorphosis. Then, XCL1 likely evolved to preferentially populate the noncanonical fold before reaching its modern-day near-equal population of folds. These discoveries illuminate how one sequence has evolved to encode multiple structures, revealing principles for protein design and engineering.

A Conserved PDZ-Binding Motif in aPKC Interacts with Par-3 and Mediates Cortical Polarity.

Par-3 regulates animal cell polarity by targeting the Par complex proteins Par-6 and atypical protein kinase C (aPKC) to specific cortical sites. Although numerous physical interactions between Par-3 and the Par complex have been identified [1-6], we discovered a novel interaction between Par-3's second PDZ domain and a highly conserved aPKC PDZ-binding motif (PBM) that is required in the context of the full-length, purified Par-6-aPKC complex. We also found that Par-3 is phosphorylated by the full Par complex and phosphorylation induces dissociation of the Par-3 phosphorylation site from aPKC's kinase domain but does not disrupt the Par-3 PDZ2-aPKC PBM interaction. In asymmetrically dividing Drosophila neuroblasts, the aPKC PBM is required for cortical targeting, consistent with its role in mediating a persistent interaction with Par-3. Our results define a physical connection that targets the Par complex to polarized sites on the cell membrane.

Asymmetric recruitment and actin-dependent cortical flows drive the neuroblast polarity cycle.

During the asymmetric divisions of Drosophila neuroblasts, the Par polarity complex cycles between the cytoplasm and an apical cortical domain that restricts differentiation factors to the basal cortex. We used rapid imaging of the full cell volume to uncover the dynamic steps that underlie transitions between neuroblast polarity states. Initially, the Par proteins aPKC and Bazooka form discrete foci at the apical cortex. Foci grow into patches that together comprise a discontinuous, unorganized structure. Coordinated cortical flows that begin near metaphase and are dependent on the actin cytoskeleton rapidly transform the patches into a highly organized apical cap. At anaphase onset, the cap disassembles as the cortical flow reverses direction toward the emerging cleavage furrow. Following division, cortical patches dissipate into the cytoplasm allowing the neuroblast polarity cycle to begin again. Our work demonstrates how neuroblasts use asymmetric recruitment and cortical flows to dynamically polarize during asymmetric division cycles.

Activation of Discs large by aPKC aligns the mitotic spindle to the polarity axis during asymmetric cell division.

Asymmetric division generates cellular diversity by producing daughter cells with different fates. In animals, the mitotic spindle aligns with Par complex polarized fate determinants, ensuring that fate determinant cortical domains are bisected by the cleavage furrow. Here, we investigate the mechanisms that couple spindle orientation to polarity during asymmetric cell division of Drosophila neuroblasts. We find that the tumor suppressor Discs large (Dlg) links the Par complex component atypical Protein Kinase C (aPKC) to the essential spindle orientation factor GukHolder (GukH). Dlg is autoinhibited by an intramolecular interaction between its SH3 and GK domains, preventing Dlg interaction with GukH at cortical sites lacking aPKC. When co-localized with aPKC, Dlg is phosphorylated in its SH3 domain which disrupts autoinhibition and allows GukH recruitment by the GK domain. Our work establishes a molecular connection between the polarity and spindle orientation machineries during asymmetric cell division.

Evolution of a Protein Interaction Domain Family by Tuning Conformational Flexibility.

Conformational flexibility allows proteins to adopt multiple functionally important conformations but can also lead to nonfunctional structures. We analyzed the dynamic behavior of the enzyme guanylate kinase as it evolved into the GK protein interaction domain (GKPID) to investigate the role of flexibility in the evolution of new protein functions. We found that the ancestral enzyme is very flexible, allowing it to adopt open conformations that can bind nucleotide and closed ones that enable catalysis of phosphotransfer from ATP to GMP. Historical mutations that converted the GK from an enzyme to a protein interaction domain dramatically reduce flexibility, predominantly by inhibiting rotations of the protein backbone that are coupled to the global closing motion. Removing flexibility prevents adoption of conformations that cannot fit the protein partner in the binding site. Our results highlight the importance of mutations that optimize protein conformational flexibility with function during evolution.

Molecular Control of Atypical Protein Kinase C: Tipping the Balance between Self-Renewal and Differentiation.

Complex organisms are faced with the challenge of generating and maintaining diverse cell types, ranging from simple epithelia to neurons and motile immune cells [1-3]. To meet this challenge, a complex set of regulatory pathways controls nearly every aspect of cell growth and function, including genetic and epigenetic programming, cytoskeleton dynamics, and protein trafficking. The far reach of cell fate specification pathways makes it particularly catastrophic when they malfunction, both during development and for tissue homeostasis in adult organisms. Furthermore, the therapeutic promise of stem cells derives from their ability to deftly navigate the multitude of pathways that control cell fate [4]. How the molecular components making up these pathways function to specify cell fate is beginning to become clear. Work from diverse systems suggests that the atypical Protein Kinase C (aPKC) is a key regulator of cell fate decisions in metazoans [5-7]. Here, we examine some of the diverse physiological outcomes of aPKC's function in differentiation, along with the molecular pathways that control aPKC and those that are responsive to changes in its catalytic activity.

Binding of Crumbs to the Par-6 CRIB-PDZ Module Is Regulated by Cdc42.

Par-6 is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. Cdc42-GTP binds the CRIB module of Par-6 and alters the binding affinity of the adjoining PDZ domain. Allosteric regulation of the Par-6 PDZ domain was first demonstrated using a peptide identified in a screen of typical carboxyl-terminal ligands. Crumbs, a membrane protein that localizes a conserved polarity complex, was subsequently identified as a functional partner for Par-6 that likely interacts with the PDZ domain. Here we show by nuclear magnetic resonance that Par-6 binds a Crumbs carboxyl-terminal peptide and report the crystal structure of the PDZ-peptide complex. The Crumbs peptide binds Par-6 more tightly than the previously studied carboxyl peptide ligand and interacts with the CRIB-PDZ module in a Cdc42-dependent manner. The Crumbs:Par-6 crystal structure reveals specific PDZ-peptide contacts that contribute to its higher affinity and Cdc42-enhanced binding. Comparisons with existing structures suggest that multiple C-terminal Par-6 ligands respond to a common conformational switch that transmits the allosteric effects of GTPase binding.

Evolution of an ancient protein function involved in organized multicellularity in animals.

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which - the evolution of GKPID's capacity to bind the cortical marker protein - can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals.

Establishment of Par-Polarized Cortical Domains via Phosphoregulated Membrane Motifs.

The Par polarity complex creates mutually exclusive cortical domains in diverse animal cells. Activity of the atypical protein kinase C (aPKC) is a key output of the Par complex as phosphorylation removes substrates from the Par domain. Here, we investigate how diverse, apparently unrelated Par substrates couple phosphorylation to cortical displacement. Each protein contains a basic and hydrophobic (BH) motif that interacts directly with phospholipids and also overlaps with aPKC phosphorylation sites. Phosphorylation alters the electrostatic character of the sequence, inhibiting interaction with phospholipids and the cell cortex. We searched for overlapping BH and aPKC phosphorylation site motifs (i.e., putative phosphoregulated BH motifs) in several animal proteomes. Candidate proteins with strong PRBH signals associated with the cell cortex but were displaced into the cytoplasm by aPKC. These findings demonstrate a potentially general mechanism for exclusion of proteins from the Par cortical domain in polarized cells.

Ordered multisite phosphorylation of lethal giant larvae by atypical protein kinase C.

In Par complex-mediated cell polarity, phosphorylation by atypical protein kinase C (aPKC) is coupled to substrate cortical displacement. Polarized substrates often contain multiple phosphorylation sites, but the role of multisite phosphorylation in Par-mediated polarity remains unclear. Here, we have dissected the role of the three aPKC phosphorylation sites within the tumor suppressor Lethal giant larvae. Using a cultured Drosophila S2 cell cortical displacement assay, we observed that phosphorylation at any one site causes only partial displacement. Complete displacement requires that all three sites be modified. We undertook a kinetic analysis to determine if aPKC phosphorylates each site equivalently. As the sites are closely spaced, we observed not only differences in the rate of phosphorylation but also interaction between the sites. A complete description of the rates reveals a preferential order of phosphorylation. Our results provide new insights into how multiple phosphorylations and phosphorylation rates could regulate localization behaviors of fate determinants at the cortex.