Modeling protein dynamics in Caenorhabditis elegans embryos reveals that the PLK-1 gradient relies on weakly coupled reaction-diffusion mechanisms.

SignificanceIntracellular gradients have essential roles in cell and developmental biology, but their formation is not fully understood. We have developed a computational approach facilitating interpretation of protein dynamics and gradient formation. We have combined this computational approach with experiments to understand how Polo-Like Kinase 1 (PLK-1) forms a cytoplasmic gradient in Caenorhabditis elegans embryos. Although the PLK-1 gradient depends on the Muscle EXcess-5/6 (MEX-5/6) proteins, we reveal differences in PLK-1 and MEX-5 gradient formation that can be explained by a model with two components, PLK-1 bound to MEX-5 and unbound PLK-1. Our combined approach suggests that a weak coupling between PLK-1 and MEX-5 reaction-diffusion mechanisms dictates the dynamic exchange of PLK-1 with the cytoplasm, explaining PLK-1 high diffusivity and smooth gradient.

PLK-1 Regulation of Asymmetric Cell Division in the Early C. elegans Embryo.

PLK1 is a conserved mitotic kinase that is essential for the entry into and progression through mitosis. In addition to its canonical mitotic functions, recent studies have characterized a critical role for PLK-1 in regulating the polarization and asymmetric division of the one-cell C. elegans embryo. Prior to cell division, PLK-1 regulates both the polarization of the PAR proteins at the cell cortex and the segregation of cell fate determinants in the cytoplasm. Following cell division, PLK-1 is preferentially inherited to one daughter cell where it acts to regulate the timing of centrosome separation and cell division. PLK1 also regulates cell polarity in asymmetrically dividing Drosophila neuroblasts and during mammalian planar cell polarity, suggesting it may act broadly to connect cell polarity and cell cycle mechanisms.

SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors.

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3'UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3'UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

Single-molecule dynamics of the P granule scaffold MEG-3 in the Caenorhabditis elegans zygote.

During the asymmetric division of the Caenorhabditis elegans zygote, germ (P) granules are disassembled in the anterior cytoplasm and stabilized/assembled in the posterior cytoplasm, leading to their inheritance by the germline daughter cell. P granule segregation depends on MEG (maternal-effect germline defective)-3 and MEG-4, which are enriched in P granules and in the posterior cytoplasm surrounding P granules. Here we use single-molecule imaging and tracking to characterize the reaction/diffusion mechanisms that result in MEG-3::Halo segregation. We find that the anteriorly enriched RNA-binding proteins MEX (muscle excess)-5 and MEX-6 suppress the retention of MEG-3 in the anterior cytoplasm, leading to MEG-3 enrichment in the posterior. We provide evidence that MEX-5/6 may work in conjunction with PLK-1 kinase to suppress MEG-3 retention in the anterior. Surprisingly, we find that the retention of MEG-3::Halo in the posterior cytoplasm surrounding P granules does not appear to contribute significantly to the maintenance of P granule asymmetry. Rather, our findings suggest that the formation of the MEG-3 concentration gradient and the segregation of P granules are two parallel manifestations of MEG-3's response to upstream polarity cues.

PIE-1 Translation in the Germline Lineage Contributes to PIE-1 Asymmetry in the Early Caenorhabditis elegans Embryo.

In the C. elegans embryo, the germline lineage is established through successive asymmetric cell divisions that each generate a somatic and a germline daughter cell. PIE-1 is an essential maternal factor that is enriched in embryonic germline cells and is required for germline specification. We estimated the absolute concentration of PIE-1::GFP in germline cells and find that PIE-1::GFP concentration increases by roughly 4.5 fold, from 92 nM to 424 nM, between the 1 and 4-cell stages. Previous studies have shown that the preferential inheritance of PIE-1 by germline daughter cells and the degradation of PIE-1 in somatic cells are important for PIE-1 enrichment in germline cells. In this study, we provide evidence that the preferential translation of maternal PIE-1::GFP transcripts in the germline also contributes to PIE-1::GFP enrichment. Through an RNAi screen, we identified Y14 and MAG-1 (Drosophila tsunagi and mago nashi) as regulators of embryonic PIE-1::GFP levels. We show that Y14 and MAG-1 do not regulate PIE-1 degradation, segregation or synthesis in the early embryo, but do regulate the concentration of maternally-deposited PIE-1::GFP. Taken together, or findings point to an important role for translational control in the regulation of PIE-1 levels in the germline lineage.

Rapid diffusion-state switching underlies stable cytoplasmic gradients in the Caenorhabditis elegans zygote.

Protein concentration gradients organize cells and tissues and commonly form through diffusion away from a local source of protein. Interestingly, during the asymmetric division of the Caenorhabditis elegans zygote, the RNA-binding proteins MEX-5 and PIE-1 form opposing concentration gradients in the absence of a local source. In this study, we use near-total internal reflection fluorescence (TIRF) imaging and single-particle tracking to characterize the reaction/diffusion dynamics that maintain the MEX-5 and PIE-1 gradients. Our findings suggest that both proteins interconvert between fast-diffusing and slow-diffusing states on timescales that are much shorter (seconds) than the timescale of gradient formation (minutes). The kinetics of diffusion-state switching are strongly polarized along the anterior/posterior (A/P) axis by the PAR polarity system such that fast-diffusing MEX-5 and PIE-1 particles are approximately symmetrically distributed, whereas slow-diffusing particles are highly enriched in the anterior and posterior cytoplasm, respectively. Using mathematical modeling, we show that local differences in the kinetics of diffusion-state switching can rapidly generate stable concentration gradients over a broad range of spatial and temporal scales.

Polo-like Kinase Couples Cytoplasmic Protein Gradients in the C. elegans Zygote.

Intracellular protein gradients underlie essential cellular and developmental processes, but the mechanisms by which they are established are incompletely understood. During the asymmetric division of the C. elegans zygote, the RNA-binding protein MEX-5 forms an anterior-rich cytoplasmic gradient that causes the RNA-binding protein POS-1 to form an opposing, posterior-rich gradient. We demonstrate that the polo-like kinase PLK-1 mediates the repulsive coupling between MEX-5 and POS-1 by increasing the mobility of POS-1 in the anterior. PLK-1 is enriched in the anterior cytoplasm and phosphorylates POS-1, which is both necessary and sufficient to increase POS-1 mobility. Regulation of POS-1 mobility depends on both the interaction between PLK-1 and MEX-5 and between MEX-5 and RNA, suggesting that MEX-5 may recruit PLK-1 to RNA in the anterior. The low concentration of MEX-5/PLK-1 in the posterior cytoplasm provides a permissive environment for the retention of POS-1, which depends on POS-1 RNA binding. Our findings describe a novel reaction/diffusion mechanism in which the asymmetric distribution of cytoplasmic PLK-1 couples two RNA-binding protein gradients, thereby partitioning the cytoplasm.

Regulation of Cell Polarity by PAR-1/MARK Kinase.

PAR-1/MARK kinases are conserved serine/threonine kinases that are essential regulators of cell polarity. PAR-1/MARK kinases localize and function in opposition to the anterior PAR proteins to control the asymmetric distribution of factors in a wide variety polarized cells. In this review, we discuss the mechanisms that control the localization and activity of PAR-1/MARK kinases, including their antagonistic interactions with the anterior PAR proteins. We focus on the role PAR-1 plays in the asymmetric division of the Caenorhabditis elegans zygote, in the establishment of the anterior/posterior axis in the Drosophila oocyte and in the control of microtubule dynamics in mammalian neurons. In addition to conserved aspects of PAR-1 biology, we highlight the unique ways in which PAR-1 acts in these distinct cell types to orchestrate their polarization. Finally, we review the connections between disruptions in PAR-1/MARK function and Alzheimer's disease and cancer.

Coupling between cytoplasmic concentration gradients through local control of protein mobility in the Caenorhabditis elegans zygote.

Cell polarity is characterized by the asymmetric distribution of factors at the cell cortex and in the cytoplasm. Although mechanisms that establish cortical asymmetries have been characterized, less is known about how persistent cytoplasmic asymmetries are generated. During the asymmetric division of the Caenorhabditis elegans zygote, the PAR proteins orchestrate the segregation of the cytoplasmic RNA-binding proteins MEX-5/6 to the anterior cytoplasm and PIE-1, POS-1, and MEX-1 to the posterior cytoplasm. In this study, we find that MEX-5/6 control the segregation of GFP::PIE-1, GFP::POS-1, and GFP::MEX-1 by locally increasing their mobility in the anterior cytoplasm. Remarkably, PIE-1, POS-1, and MEX-1 form gradients with distinct strengths, which correlates with differences in their responsiveness to MEX-5/6. We show that MEX-5/6 act downstream of the polarity regulators PAR-1 and PAR-3 and in a concentration-dependent manner to increase the mobility of GFP::PIE-1. These findings suggest that the MEX-5/6 concentration gradients are directly coupled to the establishment of posterior-rich PIE-1, POS-1, and MEX-1 concentration gradients via the formation of anterior-fast, posterior-slow mobility gradients.

Cytoplasmic localization and asymmetric division in the early embryo of Caenorhabditis elegans.

During the initial cleavages of the Caenorhabditis elegans embryo, a series of rapid and invariant asymmetric cell divisions pattern the fate, size, and position of four somatic blastomeres and a single germline blastomere. These asymmetric divisions are orchestrated by a collection of maternally deposited factors that are initially symmetrically distributed in the newly fertilized embryo. Maturation of the sperm-derived centrosome in the posterior cytoplasm breaks this symmetry by triggering a dramatic and highly stereotyped partitioning of these maternal factors. A network of conserved cell polarity regulators, the PAR proteins, form distinct anterior and posterior domains at the cell cortex. From these domains, the PAR proteins direct the segregation of somatic and germline factors into opposing regions of the cytoplasm such that, upon cell division, they are preferentially inherited by the somatic blastomere or the germline blastomere, respectively. The segregation of these factors is controlled, at least in part, by a series of reaction-diffusion mechanisms that are asymmetrically deployed along the anterior/posterior axis. The characterization of these mechanisms has important implications for our understanding of how cells are polarized and how spatial organization is generated in the cytoplasm. For further resources related to this article, please visit the WIREs website.

Regulation of the MEX-5 gradient by a spatially segregated kinase/phosphatase cycle.

Protein concentration gradients encode spatial information across cells and tissues and often depend on spatially localized protein synthesis. Here, we report that a different mechanism underlies the MEX-5 gradient. MEX-5 is an RNA-binding protein that becomes distributed in a cytoplasmic gradient along the anterior-to-posterior axis of the one-cell C. elegans embryo. We demonstrate that the MEX-5 gradient is a direct consequence of an underlying gradient in MEX-5 diffusivity. The MEX-5 diffusion gradient arises when the PAR-1 kinase stimulates the release of MEX-5 from slow-diffusive, RNA-containing complexes in the posterior cytoplasm. PAR-1 directly phosphorylates MEX-5 and is antagonized by the spatially uniform phosphatase PP2A. Mathematical modeling and in vivo observations demonstrate that spatially segregated phosphorylation and dephosphorylation reactions are sufficient to generate stable protein concentration gradients in the cytoplasm. The principles demonstrated here apply to any spatially segregated modification cycle that affects protein diffusion and do not require protein synthesis or degradation.

Domain interactions within Fzo1 oligomers are essential for mitochondrial fusion.

Mitofusins are conserved GTPases essential for the fusion of mitochondria. These mitochondrial outer membrane proteins contain a GTPase domain and two or three regions with hydrophobic heptad repeats, but little is known about how these domains interact to mediate mitochondrial fusion. To address this issue, we have analyzed the yeast mitofusin Fzo1p and find that mutation of any of the three heptad repeat regions (HRN, HR1, and HR2) leads to a null allele. Specific pairs of null alleles show robust complementation, indicating that functional domains need not exist on the same molecule. Biochemical analysis indicates that this complementation is due to Fzo1p oligomerization mediated by multiple domain interactions. Moreover, we find that two non-overlapping protein fragments, one consisting of HRN/GTPase and the other consisting of HR1/HR2, can form a complex that reconstitutes Fzo1p fusion activity. Each of the null alleles disrupts the interaction of these two fragments, suggesting that we have identified a key interaction involving the GTPase domain and heptad repeats essential for fusion.

Molecular mechanism of mitochondrial membrane fusion.

Mitochondrial fusion requires coordinated fusion of the outer and inner membranes. This process leads to exchange of contents, controls the shape of mitochondria, and is important for mitochondrial function. Two types of mitochondrial GTPases are essential for mitochondrial fusion. On the outer membrane, the fuzzy onions/mitofusin proteins form complexes in trans that mediate homotypic physical interactions between adjacent mitochondria and are likely directly involved in outer membrane fusion. Associated with the inner membrane, the OPA1 dynamin-family GTPase maintains membrane structure and is a good candidate for mediating inner membrane fusion. In yeast, Ugo1p binds to both of these GTPases to form a fusion complex, although a related protein has yet to be found in mammals. An understanding of the molecular mechanism of fusion may have implications for Charcot-Marie-Tooth subtype 2A and autosomal dominant optic atrophy, neurodegenerative diseases caused by mutations in Mfn2 and OPA1.

The WD40 protein Caf4p is a component of the mitochondrial fission machinery and recruits Dnm1p to mitochondria.

The mitochondrial division machinery regulates mitochondrial dynamics and consists of Fis1p, Mdv1p, and Dnm1p. Mitochondrial division relies on the recruitment of the dynamin-related protein Dnm1p to mitochondria. Dnm1p recruitment depends on the mitochondrial outer membrane protein Fis1p. Mdv1p interacts with Fis1p and Dnm1p, but is thought to act at a late step during fission because Mdv1p is dispensable for Dnm1p localization. We identify the WD40 repeat protein Caf4p as a Fis1p-associated protein that localizes to mitochondria in a Fis1p-dependent manner. Caf4p interacts with each component of the fission apparatus: with Fis1p and Mdv1p through its NH2-terminal half and with Dnm1p through its COOH-terminal WD40 domain. We demonstrate that mdv1delta yeast contain residual mitochondrial fission due to the redundant activity of Caf4p. Moreover, recruitment of Dnm1p to mitochondria is disrupted in mdv1delta caf4delta yeast, demonstrating that Mdv1p and Caf4p are molecular adaptors that recruit Dnm1p to mitochondrial fission sites. Our studies support a revised model for assembly of the mitochondrial fission apparatus.

Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development.

Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population.