A PDZ-kinase allosteric relay mediates Par complex regulator exchange.

The Par complex polarizes the plasma membrane of diverse animal cells using the catalytic activity of atypical PKC (aPKC) to pattern substrates. Two upstream regulators of the Par complex, Cdc42 and Par-3, bind separately to the complex to influence its activity in different ways. Each regulator binds a distinct member of the complex, Cdc42 to Par-6 and Par-3 to aPKC, making it unclear how they influence one another's binding.

Lgl resets Par complex membrane loading at mitotic exit to enable asymmetric neural stem cell division.

The Par complex regulates cell polarity in diverse animal cells , but how its localization is restricted to a specific membrane domain remains unclear. We investigated how the tumor suppressor Lethal giant larvae (Lgl) polarizes the Par complex in neural stem cells (NSCs or neuroblasts). In contrast to epithelial cells, where Lgl and the Par complex occupy mutually exclusive membrane domains, Lgl is cytoplasmic when the Par complex is apically polarized in NSCs.

Membrane oscillations driven by Arp2/3 constrict the intercellular bridge during neural stem cell divisions.

After the first furrowing step of animal cell division, the nascent sibling cells remain connected by a thin intercellular bridge (ICB). In isolated cells nascent siblings migrate away from each other to generate tension and constrict the ICB, but less is known about how cells complete cytokinesis when constrained within tissues. We examined the ICBs formed by larval brain neural stem cell (NSC) asymmetric divisions and find that they rely on constriction focused at the central midbody region rather than the flanking arms of isolated cell ICBs.

A PDZ-kinase allosteric relay mediates Par complex regulator exchange.

The Par complex polarizes the plasma membrane of diverse animal cells using the catalytic activity of atypical Protein Kinase C (aPKC) to pattern substrates. Two upstream regulators of the Par complex, Cdc42 and Par-3, bind separately to the complex to influence its activity in different ways. Each regulator binds a distinct member of the complex, Cdc42 to Par-6 and Par-3 to aPKC, making it unclear how they influence one another's binding.

The cytokinetic midbody mediates asymmetric fate specification at mitotic exit during neural stem cell division.

Asymmetric cell division (ACD) is a broadly used mechanism for generating cellular diversity. Molecules known as fate determinants are segregated during ACD to generate distinct sibling cell fates, but determinants should not be activated until fate can be specified asymmetrically. Determinants could be activated after cell division but many animal cells complete division long after mitosis ends, raising the question of how activation could occur at mitotic exit taking advantage of the unique state plasticity at this time point.

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.

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).

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.

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.

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.

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

The Par complex dynamically polarizes to the apical cortex of asymmetrically dividing 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.

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.

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.

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.

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

During the asymmetric divisions of 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.

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 neuroblasts.

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 (GK) 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.

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.

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.

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.

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.

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.