Fluorescence microscopy of actin- and microtubule-associated septins in mammalian cells.

Septins are a major component of the mammalian cytoskeleton. Septins associate with filamentous actin (F-actin) and microtubules, but the nature and significance of these interactions are not well understood. Fluorescence microscopy of F-actin- and microtubule-associated septins in fixed and living cells has been instrumental in uncovering septin functions in cellular morphogenesis and cytoskeleton-dependent processes (eg, cell division, cell migration). Here, we provide a detailed methodology for the visualization of endogenous septins by immunofluorescence microscopy, discussing sample preparation and reagents that are critical for optimal staining. In addition, we review approaches for the construction and expression of fluorescent septins and their time-lapse imaging with F-actin and microtubules. The recommended methodology is adaptable for high- and superresolution imaging of mammalian cells with various instrumentation, including wide-field and confocal microscopy as well as total internal reflection fluorescence and structured illumination microscopy.

Selective export of MHC class I molecules from the ER after their dissociation from TAP.

It has been assumed that upon dissociation from TAP, MHC class I molecules exit the ER by nonselective bulk flow. We now show that exit must occur by association with cargo receptors. Inconsistent with exit by bulk flow, loading of MHC class I molecules with high-affinity peptides triggers dissociation from TAP but has no effect on rates of ER-to-Golgi transport. Moreover, peptide-loaded MHC class I molecules accumulate at ER exit sites from which TAP molecules are excluded. Consistent with receptor-mediated exit, ER-to-Golgi transport of MHC class I molecules is independent of their cytoplasmic tails, which themselves lack ER export motifs. In addition, we show that MHC class I molecules associate with the putative cargo receptor BAP31.

Lateral diffusion of GFP-tagged H2Ld molecules and of GFP-TAP1 reports on the assembly and retention of these molecules in the endoplasmic reticulum.

Lateral diffusion of GFP-tagged H2Ld molecules in the ER membrane reports on their interaction with the TAP complex during synthesis and peptide loading. Peptide-loaded H2Ld molecules diffuse rapidly, near the theoretical limit for proteins in a bilayer. However, these molecules are retained in the ER for some time after assembly. H2Ld molecules, associated with the TAP complex, diffuse slowly, as does GFP-tagged TAP1. This implies that the association of H2Ld molecules with the TAP complex is stable for at least several minutes. It also suggests that the TAP complex is very large, perhaps containing hundreds of proteins.

Biosynthesis of Di-myo-inositol-1,1'-phosphate, a novel osmolyte in hyperthermophilic archaea.

Biosynthesis of di-myo-inositol-1,1'-phosphate (DIP) is proposed to occur with myo-inositol and myo-inositol 1-phosphate (I-1-P) used as precursors. Activation of the I-1-P with CTP and condensation of the resultant CDP-inositol (CDP-I) with myo-inositol then generates DIP. The sole known biosynthetic pathway of inositol in all organisms is the conversion of D-glucose-6-phosphate to myo-inositol. This conversion requires two key enzymes: L-I-1-P synthase and I-1-P phosphatase. Enzymatic assays using 31P nuclear magnetic resonance spectroscopy as well as a colorimetric assay for inorganic phosphate have confirmed the occurrence of L-I-1-P synthase and a moderately specific I-1-P phosphatase. The enzymatic reaction that couples CDP-I with myo-inositol to generate DIP has also been detected in Methanococcus igneus. 13C labeling studies with [2,3-13C]pyruvate and [3-13C]pyruvate were used to examine this pathway in M. igneus. Label distribution in DIP was consistent with inositol units formed from glucose-6-phosphate, but the label in the glucose moiety was scrambled via transketolase and transaldolase activities of the pentose phosphate pathway.