The self-assembling, mechanoenzymatic dynamin superfamily GTPase, dynamin-related protein 1 (Drp1), catalyzes mitochondrial and peroxisomal fission. Distinct intrinsically disordered regions (IDRs) in Drp1 substitute for the canonical pleckstrin homology (PH) domain and proline-rich domain (PRD) of prototypical dynamin, which cooperatively regulate endocytic vesicle scission. Whether the Drp1 IDRs function analogously to the corresponding dynamin domains however remains unknown. We show that an IDR unique to the Drp1 GTPase (G) domain, the ‘extended 80-loop’, albeit dissimilar in location, structure, and mechanism, functions akin to the dynamin PRD by enabling stable Drp1 mitochondrial recruitment and by suppressing Drp1 cooperative GTPase activity in the absence of specific partner-protein interactions. Correspondingly, we find that another IDR, the Drp1 variable domain (VD), in conjunction with the conserved stalk L1N loop, functions akin to the dynamin PH domain; first, in an ‘auto-inhibitory’ capacity that restricts Drp1 activity through a long-range steric inhibition of helical inter-rung G-domain dimerization, and second, as a ‘fulcrum’ for Drp1 self-assembly in the proper helical register. We show that the Drp1 VD is necessary and sufficient for specific Drp1-phospholipid interactions. We further demonstrate that the membrane-dependent VD conformational rearrangement essential for the alleviation of Drp1 auto-inhibition is contingent upon the basal GTP hydrolysis-dependent generation of Drp1 dimers from oligomers in solution. IDRs thus conformationally couple the enzymatic and membrane activities of Drp1 toward membrane fission.

Nonsense mutations are present in 10% of patients with CF, produce a premature termination codon in CFTR mRNA causing early termination of translation, and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study, we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels, demonstrates absence of CFTR function, and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly, CFTR restoration is observed in G542X intestinal organoids treated with G418, an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations.

Gastrointestinal dysfunction in Cystic Fibrosis (CF) is a prominent source of pain among patients with CF. Linaclotide, a Guanylate Cyclase C (GCC) receptor agonist, is an FDA-approved drug prescribed for chronic constipation, but has not been widely utilized in CF, as the cystic fibrosis transmembrane conductance regulator (CFTR) is the main mechanism of action. However, anecdotal clinical evidence suggests that linaclotide may be effective for treating some gastrointestinal symptoms in CF. The goal of this study was to determine the effectiveness and mechanism of linaclotide in treating CF gastrointestinal disorders utilizing CF mouse models. Intestinal transit, chloride secretion, and intestinal lumen fluidity was assessed in wildtype and CF mouse models in response to linaclotide. CFTR and Sodium/Hydrogen Exchanger 3 (NHE3) response to linaclotide was also evaluated. Linaclotide treatment improved intestinal transit in mice carrying either F508del or null Cftr mutations but did not induce detectable Cl- secretion. Linaclotide increased fluid retention and fluidity of CF intestinal contents, suggesting inhibition of fluid absorption. Targeted inhibition of sodium absorption by the NHE3 inhibitor tenapanor produced similar improvements in gastrointestinal transit as linaclotide treatment, suggesting that inhibition of fluid absorption by linaclotide contributes to improved gastrointestinal transit in CF. Our results demonstrate that linaclotide improves gastrointestinal transit in CF mouse models by increasing luminal fluidity through inhibiting NHE3 mediated sodium absorption. Further studies are necessary to assess whether linaclotide could improve CF intestinal pathologies in patients. GCC signaling and NHE3 inhibition may be therapeutic targets for CF intestinal manifestations.