The F-actin modifier villin regulates insulin granule dynamics and exocytosis downstream of islet cell autoantigen 512.

OBJECTIVE: Insulin release from pancreatic islet ß cells should be tightly controlled to avoid hypoglycemia and insulin resistance. The cortical actin cytoskeleton is a gate for regulated exocytosis of insulin secretory granules (SGs) by restricting their mobility and access to the plasma membrane. Prior studies suggest that SGs interact with F-actin through their transmembrane cargo islet cell autoantigen 512 (Ica512) (also known as islet antigen 2/Ptprn). Here we investigated how Ica512 modulates SG trafficking and exocytosis. METHODS: Transcriptomic changes in Ica512 (-/-) mouse islets were analyzed. Imaging as well as biophysical and biochemical methods were used to validate if and how the Ica512-regulated gene villin modulates insulin secretion in mouse islets and insulinoma cells. RESULTS: The F-actin modifier villin was consistently downregulated in Ica512 (-/-) mouse islets and in Ica512-depleted insulinoma cells. Villin was enriched at the cell cortex of ß cells and dispersed villin (-/-) islet cells were less round and less deformable. Basal mobility of SGs in villin-depleted cells was enhanced. Moreover, in cells depleted either of villin or Ica512 F-actin cages restraining cortical SGs were enlarged, basal secretion was increased while glucose-stimulated insulin release was blunted. The latter changes were reverted by overexpressing villin in Ica512-depleted cells, but not vice versa. CONCLUSION: Our findings show that villin controls the size of the F-actin cages restricting SGs and, thus, regulates their dynamics and availability for exocytosis. Evidence that villin acts downstream of Ica512 also indicates that SGs directly influence the remodeling properties of the cortical actin cytoskeleton for tight control of insulin secretion.

Molecular genetic analysis of pollen irradiation mutagenesis in Arabidopsis.

• Pollen γ-irradiation mutagenesis was systematically investigated to generate targeted mutations in Arabidopsis. • Irradiation effects on viability, germination and frequency of loci deletions were evaluated. • Mutation frequency increased with irradiation dose and varied depending upon pollen developmental stage. Meiosis was the most irradiation-sensitive stage, however, it did not equate to the highest mutation frequency. High frequencies of targeted mutations were obtained by irradiation from the second mitosis to mature pollen stages, using 400-600 Gy. Targeted mutations could also be obtained using lower doses of γ-rays (e.g. 200 Gy) provided that pollen was irradiated at an earlier developmental stage. ms1ttg marker locus pseudo-dominants were used to verify the presence and size of the resultant deletions. • The results demonstrate that γ-irradiation of pollen is an efficient approach for generating deletions in the Arabidopsis genome. Pollen mutagenesis offers the possibility of combining single-cell selection procedures with the advantages of haploid systems, including the ability to treat large numbers of pollen grains, the absence of chimerism and direct expression of targeted alleles in the M1 generation.

Age-dependent labeling and imaging of insulin secretory granules.

Insulin is stored within the secretory granules of pancreatic ß-cells, and impairment of its release is the hallmark of type 2 diabetes. Preferential exocytosis of newly synthesized insulin suggests that granule aging is a key factor influencing insulin secretion. Here, we illustrate a technology that enables the study of granule aging in insulinoma cells and ß-cells of knock-in mice through the conditional and unequivocal labeling of insulin fused to the SNAP tag. This approach, which overcomes the limits encountered with previous strategies based on radiolabeling or fluorescence timer proteins, allowed us to formally demonstrate the preferential release of newly synthesized insulin and reveal that the motility of cortical granules significantly changes over time. Exploitation of this approach may enable the identification of molecular signatures associated with granule aging and unravel possible alterations of granule turnover in diabetic ß-cells. Furthermore, the method is of general interest for the study of membrane traffic and aging.

Human vaccine research in the European Union.

The use of vaccines is saving millions of lives every year across the globe, but a number of important diseases such as HIV/AIDS, malaria, TB and hepatitis C continue to frustrate attempts to produce effective vaccines against them. Confronting these challenges will require new approaches and increased research efforts by the scientific community. The Sixth Framework Programme (FP6; 2002-2006) of the European Commission (EC) has been an important catalyst in this direction by allocating a financial contribution of more than EUR 210 million to a wide variety of vaccine research activities, ranging from basic vaccinology, translational research to clinical application of vaccines. Taken together, around 581 research groups from 52 countries are participating in the vaccine activities of FP6. This impressive number signals a new spirit of collaborative research, which will facilitate the exploitation of the immense possibilities in modern vaccinology.