Real-Time Measurement of F-Actin Remodelling during Exocytosis Using Lifeact-EGFP Transgenic Animals

F-actin remodelling is essential for a wide variety of cell processes. It is important in exocytosis, where F-actin coats fusing exocytic granules. The purpose of these F-actin coats is unknown. They may be important in stabilizing the fused granules, they may play a contractile role and promote expulsion of granule content and finally may be important in endocytosis. To elucidate these functions of F-actin remodelling requires a reliable method to visualize F-actin dynamics in living cells. The recent development of Lifeact-EGFP transgenic animals offers such an opportunity. Here, we studied the characteristics of exocytosis in pancreatic acinar cells obtained from the Lifeact-EGFP transgenic mice. We show that the time-course of agonist-evoked exocytic events and the kinetics of each single exocytic event are the same for wild type and Lifeact-EGFP transgenic animals. We conclude that Lifeact-EGFP animals are a good model to study of exocytosis and reveal that F-actin coating is dependent on the de novo synthesis of F-actin and that development of actin polymerization occurs simultaneously in all regions of the granule. Our insights using the Lifeact-EGFP mice demonstrate that F-actin coating occurs after granule fusion and is a granule-wide event

Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes

Mutations in the Catalytic Loop HRD Motif Alter the Activity and Function of Drosophila Src64

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele

Interaction of microtubules and actin during the post-fusion phase of exocytosis

Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis

FcRn-mediated antibody transport across epithelial cells revealed by electron tomography

The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers, thereby providing the fetus or newborn with humoral immunity before its immune system is fully functional. In newborn rats, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. The pH difference between the apical (pH 6.0–6.5) and basolateral (pH 7.4) sides of intestinal epithelial cells facilitates the efficient unidirectional transport of IgG, because FcRn binds IgG at pH 6.0–6.5 but not at pH 7 or more. As milk passes through the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum and jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum). Here we use electron tomography to make jejunal transcytosis visible directly in space and time, developing new labelling and detection methods to map individual nanogold-labelled Fc within transport vesicles and simultaneously to characterize these vesicles by immunolabelling. Combining electron tomography with a nonperturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine whether a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moves through networks of entangled tubular and irregular vesicles, only some of which are microtubule-associated, as it migrates to the basolateral surface. New features of transcytosis are elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis through clathrin-coated pits. Markers for early, late and recycling endosomes each labelled vesicles in different and overlapping morphological classes, revealing spatial complexity in endo-lysosomal trafficking

Differential Cerebral Cortex Transcriptomes of Baboon Neonates Consuming Moderate and High Docosahexaenoic Acid Formulas

BACKGROUND: Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) are the major long chain polyunsaturated fatty acids (LCPUFA) of the central nervous system (CNS). These nutrients are present in most infant formulas at modest levels, intended to support visual and neural development. There are no investigations in primates of the biological consequences of dietary DHA at levels above those present in formulas but within normal breastmilk levels. METHODS AND FINDINGS: Twelve baboons were divided into three formula groups: Control, with no DHA-ARA; “L”, LCPUFA, with 0.33%DHA-0.67%ARA; “L3”, LCPUFA, with 1.00%DHA-0.67%ARA. All the samples are from the precentral gyrus of cerebral cortex brain regions. At 12 weeks of age, changes in gene expression were detected in 1,108 of 54,000 probe sets (2.05%), with most showing <2-fold change. Gene ontology analysis assigns them to diverse biological functions, notably lipid metabolism and transport, G-protein and signal transduction, development, visual perception, cytoskeleton, peptidases, stress response, transcription regulation, and 400 transcripts having no defined function. PLA2G6, a phospholipase recently associated with infantile neuroaxonal dystrophy, was downregulated in both LCPUFA groups. ELOVL5, a PUFA elongase, was the only LCPUFA biosynthetic enzyme that was differentially expressed. Mitochondrial fatty acid carrier, CPT2, was among several genes associated with mitochondrial fatty acid oxidation to be downregulated by high DHA, while the mitochondrial proton carrier, UCP2, was upregulated. TIMM8A, also known as deafness/dystonia peptide 1, was among several differentially expressed neural development genes. LUM and TIMP3, associated with corneal structure and age-related macular degeneration, respectively, were among visual perception genes influenced by LCPUFA. TIA1, a silencer of COX2 gene translation, is upregulated by high DHA. Ingenuity pathway analysis identified a highly significant nervous system network, with epidermal growth factor receptor (EGFR) as the outstanding interaction partner. CONCLUSIONS: These data indicate that LCPUFA concentrations within the normal range of human breastmilk induce global changes in gene expression across a wide array of processes, in addition to changes in visual and neural function normally associated with formula LCPUFA