Erika Beyrent, Derek T Wei*, Gwendolyn M Beacham*, Sangwoo Park, Jian Zheng, Matthew J Paszek, Gunther Hollopeter. Dimerization activates the Inversin complex in C. elegans (submitted and posted 5/18/2024). *contributed equally.
Relevance: There is strong genetic and imaging data supporting the existence of an Inversin complex consisting of three proteins. In humans, loss-of-function mutations in INVS, ANKS6, and NEK8 present with the same cystic kidney disease, and all three proteins colocalize in the proximal shaft of primary cilia. In C. elegans, loss of any one of the worm homologs (MLT-4, MLT-2, and NEKL-2, respectively) cause a lethal molting defect, and the proteins colocalize at apical epidermal junctions. It has proven difficult to determine how these conserved components appear to be acting together, either as a static or dynamic assembly.
In this manuscript, we describe our discovery that the Inversin complex dimerizes to become active in C. elegans. We found that tagging MLT-4 with the red fluorescent protein, TagRFP-T, causes worms to exhibit a gain-of-function, cyst-like phenotype we affectionately call ‘jowls.’ We show that the MLT-4::RFP allele is hypermorphic, indicating that the jowls phenotype represents a simple bioassay for a constitutively active Inversin complex. To understand how the fluorescent tag induces hyperactivity, we performed a chemical-mutagenesis screen for worms lacking jowls. We isolated mutations in residues at the interface of two TagRFP-T molecules in the crystal structure, suggesting that the fluorescent tag is promoting dimerization of the Inversin complex. Single-molecule imaging of C. elegans lysates supports this model — a mutation in TagRFP-T increases the monomer fraction of MLT-4::RFP. To confirm that the active state of the Inversin complex is dimeric, we used optogenetics to show that dimerization of either MLT-4 or the kinase member of the complex (NEKL-2) is sufficient to cause jowls. Finally, we identify a lethal mutation in MLT-4 from our screen that specifically counteracts dimerization — optogenetic stimulation of NEKL-2 restores viability — demonstrating that the Inversin complex requires dimerization for function.
Our findings present the first molecular mechanism for how the Inversin complex functions. Although the Inversin complex is clearly required for proper metazoan development and physiology it has been difficult to glean mechanistic insights about the complex through loss-of-function studies of lethal mutations. Our gain-of-function studies offer a new route for determining the assembly, regulation, and potential downstream targets of the Inversin complex. More broadly, it is thought that many membrane-associated kinase complexes require dimerization for activity, and the Inversin complex appears to share this feature. Cell biologists will also be interested by our observation of the jowls phenotype induced by the TagRFP-T tag. This represents a robust assay for the propensity of any genetically encoded tag to dimerize in vivo, coupled with a simple screening strategy for fluorescent variants with improved monomeric character.
Edward A Partlow, Kevin S Cannon, Gunther Hollopeter*, Richard W Baker*. Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor complex for cargo internalization (2022). Nat. Struct. Mol. Biol. *co-corresponding authors.
Gwendolyn M Beacham, Derek T Wei*, Erika Beyrent*, Ying Zhang, Jian Zheng, Mari MK Camacho, Laurence Florens, Gunther Hollopeter. The C. elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator (2022). Genetics. *contributed equally.
first posted to bioRxiv on July 13, 2019
Gwendolyn M Beacham, Edward A Partlow, Gunther Hollopeter. Conformational regulation of AP1 and AP2 clathrin adaptor complexes (2019). Traffic 10:741-751.
Edward A Partlow, Richard W Baker*, Gwendolyn M Beacham, Joshua S Chappie, Andres E Leschziner*, Gunther Hollopeter*. A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex (2019). eLife 8:e50003. *co-corresponding authors.
first posted to bioRxiv on July 13, 2019
Gwendolyn M Beacham, Edward A Partlow, Jeffrey J Lange, Gunther Hollopeter. NECAPs are negative regulators of the clathrin adaptor complex (2018). eLife 10.7554/eLife.32242.
first posted to bioRxiv on September 22, 2017
SELECTED PUBLICATIONS FROM GUNTHER'S TRAINING:
Hollopeter G, Lange JJ, Zhang Y, Vu TV, Gu M, Ailion M, Lambie EJ, Slaughter BD, Unruh JR, Florens L, Jorgensen EM. The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex (2014). eLife 10.7554/eLife.03648.
Liu Q, Hollopeter G, Jorgensen EM. Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction (2009). Proc. Natl. Acad. Sci. 106:10823-8.
Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D, Conley PB. Identification of the platelet ADP receptor targeted by antithrombotic drugs (2001). Nature 409: 202-207.
Erickson JC, Hollopeter G, Palmiter RD. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y (1996). Science 274:1704-1707.
Relevance: There is strong genetic and imaging data supporting the existence of an Inversin complex consisting of three proteins. In humans, loss-of-function mutations in INVS, ANKS6, and NEK8 present with the same cystic kidney disease, and all three proteins colocalize in the proximal shaft of primary cilia. In C. elegans, loss of any one of the worm homologs (MLT-4, MLT-2, and NEKL-2, respectively) cause a lethal molting defect, and the proteins colocalize at apical epidermal junctions. It has proven difficult to determine how these conserved components appear to be acting together, either as a static or dynamic assembly.
In this manuscript, we describe our discovery that the Inversin complex dimerizes to become active in C. elegans. We found that tagging MLT-4 with the red fluorescent protein, TagRFP-T, causes worms to exhibit a gain-of-function, cyst-like phenotype we affectionately call ‘jowls.’ We show that the MLT-4::RFP allele is hypermorphic, indicating that the jowls phenotype represents a simple bioassay for a constitutively active Inversin complex. To understand how the fluorescent tag induces hyperactivity, we performed a chemical-mutagenesis screen for worms lacking jowls. We isolated mutations in residues at the interface of two TagRFP-T molecules in the crystal structure, suggesting that the fluorescent tag is promoting dimerization of the Inversin complex. Single-molecule imaging of C. elegans lysates supports this model — a mutation in TagRFP-T increases the monomer fraction of MLT-4::RFP. To confirm that the active state of the Inversin complex is dimeric, we used optogenetics to show that dimerization of either MLT-4 or the kinase member of the complex (NEKL-2) is sufficient to cause jowls. Finally, we identify a lethal mutation in MLT-4 from our screen that specifically counteracts dimerization — optogenetic stimulation of NEKL-2 restores viability — demonstrating that the Inversin complex requires dimerization for function.
Our findings present the first molecular mechanism for how the Inversin complex functions. Although the Inversin complex is clearly required for proper metazoan development and physiology it has been difficult to glean mechanistic insights about the complex through loss-of-function studies of lethal mutations. Our gain-of-function studies offer a new route for determining the assembly, regulation, and potential downstream targets of the Inversin complex. More broadly, it is thought that many membrane-associated kinase complexes require dimerization for activity, and the Inversin complex appears to share this feature. Cell biologists will also be interested by our observation of the jowls phenotype induced by the TagRFP-T tag. This represents a robust assay for the propensity of any genetically encoded tag to dimerize in vivo, coupled with a simple screening strategy for fluorescent variants with improved monomeric character.
Edward A Partlow, Kevin S Cannon, Gunther Hollopeter*, Richard W Baker*. Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor complex for cargo internalization (2022). Nat. Struct. Mol. Biol. *co-corresponding authors.
Gwendolyn M Beacham, Derek T Wei*, Erika Beyrent*, Ying Zhang, Jian Zheng, Mari MK Camacho, Laurence Florens, Gunther Hollopeter. The C. elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator (2022). Genetics. *contributed equally.
first posted to bioRxiv on July 13, 2019
Gwendolyn M Beacham, Edward A Partlow, Gunther Hollopeter. Conformational regulation of AP1 and AP2 clathrin adaptor complexes (2019). Traffic 10:741-751.
Edward A Partlow, Richard W Baker*, Gwendolyn M Beacham, Joshua S Chappie, Andres E Leschziner*, Gunther Hollopeter*. A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex (2019). eLife 8:e50003. *co-corresponding authors.
first posted to bioRxiv on July 13, 2019
Gwendolyn M Beacham, Edward A Partlow, Jeffrey J Lange, Gunther Hollopeter. NECAPs are negative regulators of the clathrin adaptor complex (2018). eLife 10.7554/eLife.32242.
first posted to bioRxiv on September 22, 2017
SELECTED PUBLICATIONS FROM GUNTHER'S TRAINING:
Hollopeter G, Lange JJ, Zhang Y, Vu TV, Gu M, Ailion M, Lambie EJ, Slaughter BD, Unruh JR, Florens L, Jorgensen EM. The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex (2014). eLife 10.7554/eLife.03648.
Liu Q, Hollopeter G, Jorgensen EM. Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction (2009). Proc. Natl. Acad. Sci. 106:10823-8.
Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D, Conley PB. Identification of the platelet ADP receptor targeted by antithrombotic drugs (2001). Nature 409: 202-207.
Erickson JC, Hollopeter G, Palmiter RD. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y (1996). Science 274:1704-1707.