Harterink M et al. (2016) Curr Biol "Light-controlled intracellular transport in Caenorhabditis elegans."

Harterink M, van Bergeijk P, Allier C, Hoogenraad CC, de Haan B, van den Heuvel S, Kapitein LC

[Curr Biol, 2016]

To establish and maintain their complex morphology and function, neurons and other polarized cells exploit cytoskeletal motor proteins to distribute cargoes to specific compartments [1]. Recent studies in cultured cells have used inducible motor protein recruitment to explore how different motors contribute to polarized transport and to control the subcellular positioning of organelles [2,3]. Such approaches also seem promising avenues for studying motor activity and organelle positioning within more complex cellular assemblies, but their applicability to multicellular in vivo systems has so far remained unexplored. Here, we report the development of an optogenetic organelle transport strategy in the in vivo model system Caenorhabditis elegans. We demonstrate that movement and pausing of various organelles can be achieved by recruiting the proper cytoskeletal motor protein with light. In neurons, we find that kinesin and dynein exclusively target the axon and dendrite, respectively, revealing the basic principles for polarized transport. In vivo control of motor attachment and organelle distributions will be widely useful in exploring the mechanisms that govern the dynamic morphogenesis of cells and tissues, within the context of a developing animal.

Harterink M et al. (2017) Nat Methods "DeActs: genetically encoded tools for perturbing the actin cytoskeleton in single cells."

Lang AE, Zuchero JB, Will L, Hoogenraad CC, Harterink M, Turan J, Ibrahim A, Pasterkamp RJ, Kapitein LC, da Silva ME, Kudryashov D, van Battum EY, Barres BA

[Nat Methods, 2017]

The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively induce actin disassembly in eukaryotic cells. We demonstrate that DeActs are universal tools for studying the actin cytoskeleton in single cells in culture, tissues, and multicellular organisms including various neurodevelopmental model systems.

Harterink M et al. (2018) J Cell Sci "Local microtubule organization promotes cargo transport in C. elegans dendrites."

Yau KW, Harterink M, Kapitein LC, de Haan B, Miller KG, van den Heuvel S, Edwards SL, Hoogenraad CC

[J Cell Sci, 2018]

The specific organization of the neuronal microtubule cytoskeleton in axons and dendrites is an evolutionarily conserved determinant of neuronal polarity, allowing for selective cargo sorting. However, how dendritic microtubules are organized and whether local differences influence cargo transport remains largely unknown. Here, we use live-cell imaging to systematically probe the microtubule organization in <i>C. elegans</i> neurons and demonstrate the contribution of distinct mechanisms in the organization of dendritic microtubules. We found that most non-ciliated neurons depend on <i>unc-116</i> (kinesin-1), <i>unc-33</i> (CRMP) and <i>unc-44</i> (ankyrin) for proper microtubule organization and polarized cargo transport, as previously reported. Ciliated neurons and the URX neuron however, use an additional pathway to nucleate microtubules from the tip of the dendrite, at the base of the sensory cilium in ciliated neurons. Since, inhibition of distal microtubule nucleation affects distal dendritic transport, we propose a model in which the presence of a microtubule organizing center at the dendrite tip ensures proper dendritic cargo transport.

Harterink M et al. (2011) Development "Neuroblast migration along the anteroposterior axis of C. elegans is controlled by opposing gradients of Wnts and a secreted Frizzled-related protein."

Middelkoop TC, Kim DH, Korswagen HC, Doan TD, van Oudenaarden A, Harterink M

[Development, 2011]

The migration of neuroblasts along the anteroposterior body axis of C. elegans is controlled by multiple Wnts that act partially redundantly to guide cells to their precisely defined final destinations. How positional information is specified by this system is, however, still largely unknown. Here, we used a novel fluorescent in situ hybridization methods to generate a quantitative spatiotemporal expression map of the C. elegans Wnt genes. We found that the five Wnt genes are expressed in a series of partially overlapping domains along the anteroposterior axis, with a predominant expression in the posterior half of the body. Furthermore, we show that a secreted Frizzled-related protein is expressed at the anterior end of the body axis, where it inhibits Wnt signaling to control neuroblast migration. Our findings reveal that a system of regionalized Wnt gene expression and anterior Wnt inhibition guides the highly stereotypic migration of neuroblasts in C. elegans. Opposing expression of Wnts and Wnt inhibitors has been observed in basal metazoans and in the vertebrate neurectoderm. Our results in C. elegans support the notion that a system of posterior Wnt signaling and anterior Wnt inhibition is an evolutionarily conserved principle of primary body axis specification.

Harterink M et al. (2011) Nat Cell Biol "A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion."

van Heesbeen RG, Basler K, Betist MC, Korswagen HC, McGough IJ, Cullen PJ, Middelkoop TC, van Weering JR, Port F, Lorenowicz MJ, Silhankova M, Harterink M

[Nat Cell Biol, 2011]

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.

Harterink, M. et al. (2009) International Worm Meeting "The Myotubularin complex MTM-6/MTM-9 and the PX domain containing protein SNX-3 play a crucial role in MIG-14/Wls recycling in Wnt producing cells."

Korswagen, H.C., Betist, M., Silhankova, M., Harterink, M., van Heesbeen, R.G.H.P.

[International Worm Meeting, 2009]

The Wnt family of secreted signaling proteins is responsible for important developmental and homeostatic processes throughout the animal kingdom. Furthermore, deregulation of Wnt signaling is implicated in several human pathologies, most notably cancer. Although much is known about how the Wnt signal is transduced into different intracellular responses, much less is known about how a functional Wnt protein is produced and secreted. MIG-14/Wntless (Wls), a conserved multi-pass transmembrane protein, can bind Wnt and is required for its secretion. It is proposed to function as a Wnt sorting receptor, transporting Wnt from the Golgi to the plasma membrane. After Wnt release, MIG-14/Wls is recycled back to the Golgi by the retromer, a protein complex required for endosome to Golgi retrograde transport of sorting receptors. In order to better understand this mechanism, we performed a genome wide RNAi screen in different sensitized genetic backgrounds for genes that are required for signaling by the Wnt EGL-20. In this screen, we identified several genes involved in phosphatidylinositol 3-phosphate (PI3P) turnover. This lipid is enriched on endosomal membranes and is important for trafficking, since it recruits key regulators of intracellular transport. We found that mutation of one of the Myotubularin PI3P phosphatase complexes, MTM-6/MTM-9, leads to defects in Wnt signaling. Since mutation or knock down of the other Myotubularins does not significantly affect Wnt signaling, the MTM-6/9 complex may regulate a specific pool of PI3P that is required for Wnt signaling. The mtm-6/9 Wnt phenotype can be rescued by knock down of components of the PI 3-kinase complex, showing the importance of a correct PI3P balance. Membrane tethering of the core-retromer is thought to be mediated via members of the sorting nexin family, which can bind PI3P through a PX domain. Surprisingly, however, mutants for the classical retromer sorting nexins snx-1 and snx-5 do not show any Wnt signaling defects. In the screen we identified another sorting nexin, snx-3. The snx-3 mutant shows strong Wnt signaling defects, which are similar but not identical to retromer mutants. We found that both mtm-6 and snx-3 are required in the Wnt producing cells, and that both are required for proper recycling of MIG-14/Wls. Furthermore, we found that depletion of mtm-6 affects the cellular localization of SNX-3. Therefore, we propose that the MTM-6/9 complex regulates the recruitment of SNX-3 to endosomes, which in turn enables the retromer to recycle MIG-14/Wls.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Ramalho, J.J. et al. (2017) International Worm Meeting "Regulation of ERM proteins in cortical membrane remodeling."

Ramalho, J.J., Boxem, M., Harterink, M.

[International Worm Meeting, 2017]

Dynamic remodeling of specific cortical domains occurs in most polarized cells and is an essential process for animal development and body homeostasis. Membrane remodeling requires interaction between the plasma membrane and the underlying cytoskeletal actin network. Proteins of the conserved Ezrin-Radixin-Moesin (ERM) family interact with membrane components through an N-terminal FERM domain as well as with F-actin via the C-terminal domain. In different model systems, ERM linker activity has been associated with formation of dynamic actin-based structures such as microvilli, axonal growth cones, or the leading edge of migratory cells. Activity of ERM proteins depends on a conformational change that turns an inactive cytoplasmic form into an active membrane- and actin-bound form. In vitro data suggests an activation model dependent on PIP2-binding followed by phosphorylation of a conserved C-terminal residue. However, in vivo data supporting this model is scarce and contradictory. We use the single C. elegans ERM ortholog, erm-1, as a model to study the contribution of different regulatory sites for ERM protein activity and tissue morphogenesis in vivo. Using CRISPR/Cas9 gene editing and quantitative live-cell imaging we show that PIP2-binding, but not phosphorylation of the C-terminal T544 residue, is critically required for ERM-1 function. erm-1 mutants unable to bind PIP2 mimic the null phenotype. In contrast, mutants that either constitutively lack or mimic T544 phosphorylation are viable, with defects whose severity differs between tissues. Both erm-1(T544A) and erm-1(T544D) mutants show severe defects in excretory canal development, and our data suggests that ERM-1 T544 phosphocycling is necessary for canal lumenogenesis. In the intestine, phosphocycling appears to regulate ERM-1 stability and trafficking of apical cargo. However, the effects of T544A and T544D mutations on the apical actin ACT-5 differ. Expression of the unphosphorylatable ERM-1 T544A variant decreases the levels and stability of ACT-5, while the phosphomimicking ERM-1 T544D mutation induces formation of additional luminal membrane, but does not affect ACT-5 levels or stability. We are currently investigating how these mutations affect ERM-1 dynamics, and how they affect the intestine at the ultrastructural level. Unraveling the regulatory mechanisms behind ERM protein function will contribute to our understanding of membrane remodeling events during development and disease.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[J Infect Dis, 2015]

BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and &gt;100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.