A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures.

CCDC39 and CCDC40 were first identified as causative mutations in primary ciliary dyskinesia patients; cilia from patients show disorganized microtubules, and they are missing both N-DRC and inner dynein arms proteins. In Chlamydomonas, we used immunoblots and microtubule sliding assays to show that mutants in CCDC40 (PF7) and CCDC39 (PF8) fail to assemble N-DRC, several inner dynein arms, tektin, and CCDC39. Enrichment screens for suppression of pf7; pf8 cells led to the isolation of five independent extragenic suppressors defined by four different mutations in a NIMA-related kinase, CNK11. These alleles partially rescue the flagellar length defect, but not the motility defect. The suppressor does not restore the missing N-DRC and inner dynein arm proteins. In addition, the cnk11 mutations partially suppress the short flagella phenotype of N-DRC and axonemal dynein mutants, but do not suppress the motility defects. The tpg1 mutation in TTLL9, a tubulin polyglutamylase, partially suppresses the length phenotype in the same axonemal dynein mutants. In contrast to cnk11, tpg1 does not suppress the short flagella phenotype of pf7. The polyglutamylated tubulin in the proximal region that remains in the tpg1 mutant is reduced further in the pf7; tpg1 double mutant by immunofluorescence. CCDC40, which is needed for docking multiple other axonemal complexes, is needed for tubulin polyglutamylation in the proximal end of the flagella. The CCDC39 and CCDC40 proteins are likely to be involved in recruiting another tubulin glutamylase(s) to the flagella. Another difference between cnk11-1 and tpg1 mutants is that cnk11-1 cells show a faster turnover rate of tubulin at the flagellar tip than in wild-type flagella and tpg1 flagella show a slower rate. The double mutant shows a turnover rate similar to tpg1, which suggests the faster turnover rate in cnk11-1 flagella requires polyglutamylation. Thus, we hypothesize that many short flagella mutants in Chlamydomonas have increased instability of axonemal microtubules. Both CNK11 and tubulin polyglutamylation play roles in regulating the stability of axonemal microtubules.

Single-image diffusion coefficient measurements of proteins in free solution.

Diffusion coefficient measurements are important for many biological and material investigations, such as studies of particle dynamics and kinetics, and size determinations. Among current measurement methods, single particle tracking (SPT) offers the unique ability to simultaneously obtain location and diffusion information about a molecule while using only femtomoles of sample. However, the temporal resolution of SPT is limited to seconds for single-color-labeled samples. By directly imaging three-dimensional diffusing fluorescent proteins and studying the widths of their intensity profiles, we were able to determine the proteins' diffusion coefficients using single protein images of submillisecond exposure times. This simple method improves the temporal resolution of diffusion coefficient measurements to submilliseconds, and can be readily applied to a range of particle sizes in SPT investigations and applications in which diffusion coefficient measurements are needed, such as reaction kinetics and particle size determinations.

Accuracy of retrograde ileostomy radiographic examination for detecting small-bowel abnormalities.

OBJECTIVE: The purpose of this study was to assess the accuracy of retrograde ileostomy radiographic examination for detecting small-bowel abnormalities in patients with ileostomies. MATERIALS AND METHODS: Our database contained the records of 35 patients who had undergone retrograde ileostomy examinations from 1999-2005 and fulfilled our inclusion criteria. The images from the examinations were reviewed to determine the frequency and nature of small-bowel abnormalities, whether lateral views were obtained, and whether the catheter tip was withdrawn to the stoma. The accuracy of these examinations was determined by correlating clinical, radiographic, and surgical findings. RESULTS: Twenty-eight (80%) of the patients had small-bowel abnormalities: adhesions in 14 (40%), adynamic small-bowel ileus in four (11%), strictures in two (6%), Crohn's disease in two (6%), focal perforation in two (6%), fistulas (one enterocutaneous and one enterovaginal) in two (6%), metastasis in one (3%), and parastomal hernia in one (3%) of the patients. The other seven (20%) patients had normal findings. Abnormalities were detected on lateral but not frontal or oblique views in six (75%) of eight patients with distal ileal disease and in three (43%) of seven patients for whom views were obtained only after withdrawal of the catheter to the stoma. Retrograde ileostomy examination had a sensitivity of 96%, specificity of 86%, positive predictive value of 96%, and negative predictive value of 86%. CONCLUSION: Our experience suggested that retrograde ileostomy examination is an accurate technique for detecting symptomatic small-bowel abnormalities in patients with ileostomies, particularly in the distal-most portion of the ileum abutting the ileostomy stoma.