Dimerization activates the Inversin complex in C. elegans.

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through characterization of hyperactive alleles in C. elegans , we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologs of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states-an active dimer and an inactive monomer-gates the output of the Inversin complex.

Complement-dependent loss of inhibitory synapses on pyramidal neurons following Toxoplasma gondii infection.

The apicomplexan parasite Toxoplasma gondii has developed mechanisms to establish a central nervous system infection in virtually all warm-blooded animals. Acute T. gondii infection can cause neuroinflammation, encephalitis, and seizures. Meanwhile, studies in humans, nonhuman primates, and rodents have linked chronic T. gondii infection with altered behavior and increased risk for neuropsychiatric disorders, including schizophrenia. These observations and associations raise questions about how this parasitic infection may alter neural circuits. We previously demonstrated that T. gondii infection triggers the loss of inhibitory perisomatic synapses, a type of synapse whose dysfunction or loss has been linked to neurological and neuropsychiatric disorders. We showed that phagocytic cells (including microglia and infiltrating monocytes) contribute to the loss of these inhibitory synapses. Here, we show that these phagocytic cells specifically ensheath excitatory pyramidal neurons, leading to the preferential loss of perisomatic synapses on these neurons and not those on cortical interneurons. Moreover, we show that infection induces an increased expression of the complement C3 gene, including by populations of these excitatory neurons. Infecting C3-deficient mice with T. gondii revealed that C3 is required for the loss of perisomatic inhibitory synapses. Interestingly, loss of C1q did not prevent the loss of perisomatic synapses following infection. Together, these findings provide evidence that T. gondii induces changes in excitatory pyramidal neurons that trigger the selective removal of inhibitory perisomatic synapses and provide a role for a nonclassical complement pathway in the remodeling of inhibitory circuits in the infected brain.

The Caenorhabditis elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator.

How serine/threonine phosphatases are spatially and temporally tuned by regulatory subunits is a fundamental question in cell biology. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins are protein phosphatase 1 catalytic subunit binding partners associated with cardiocutaneous diseases. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins localize protein phosphatase 1 catalytic subunit to cell-cell junctions, but how ankyrin repeat, SH3 domain, proline-rich-region-containing proteins localize and whether they regulate protein phosphatase 1 catalytic subunit activity in vivo is unclear. Through a Caenorhabditis elegans genetic screen, we find that loss of the ankyrin repeat, SH3 domain, proline-rich-region-containing protein homolog, APE-1, suppresses a pathology called "jowls," providing us with an in vivo assay for APE-1 activity. Using immunoprecipitations and mass spectrometry, we find that APE-1 binds the protein phosphatase 1 catalytic subunit called GSP-2. Through structure-function analysis, we discover that APE-1's N-terminal half directs the APE-1-GSP-2 complex to intercellular junctions. Additionally, we isolated mutations in highly conserved residues of APE-1's ankyrin repeats that suppress jowls yet do not preclude GSP-2 binding, implying APE-1 does more than simply localize GSP-2. Indeed, in vivo reconstitution of APE-1 suggests the ankyrin repeats modulate phosphatase output, a function we find to be conserved among vertebrate homologs.

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment.

The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214-218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.

An N-terminal Flag-tag impairs TPP1 regulation of telomerase function.

The shelterin protein complex protects natural chromosome ends from being recognized as DNA damage sites and also regulates the synthesis of telomeric repeats by telomerase. TPP1, a shelterin subunit that is essential for telomerase extension of telomeres, has been studied intensively in recent years. Many such studies utilize epitope tagged TPP1, but it is unclear how the tags may affect the multiple cellular functions of TPP1. Here we analyzed the effect of adding a 3x Flag epitope tag to the N- or C-terminus of TPP1. While the position of the tag did not affect TPP1's interaction within the shelterin complex or its localization to telomeres, the N-terminal Flag tag on TPP1 impaired telomerase function, resulting in reduced telomerase processivity in vitro and a failure to stimulate telomere elongation in vivo. The C-terminally Flag-tagged TPP1, in contrast, behaved similarly to untagged TPP1 in all functional aspects examined. These findings suggest that caution is required when utilizing epitope tagged TPP1 to study its regulation of telomerase function.