Connexin 43 Functions as a Positive Regulator of Stem Cell Differentiation into Definitive Endoderm and Pancreatic Progenitors.

Efficient stem cell differentiation into pancreatic islet cells is of critical importance for the development of cell replacement therapies for diabetes. Here, we identify the expression pattern of connexin 43 (Cx43), a gap junction (GJ) channel protein, in human embryonic stem cell (hESC)-derived definitive endoderm (DE) and primitive gut tube cells, representing early lineages for posterior foregut (PF), pancreatic progenitors (PP), pancreatic endocrine progenitors (PE), and islet cells. As the function of GJ channels is dependent on their gating status, we tested the impact of supplementing hESC-derived PP cell cultures with AAP10, a peptide that promotes Cx43 GJ channel opening.

Determining the number of specific proteins in cellular compartments by quantitative microscopy.

This protocol describes a method for determining both the average number and variance of proteins, in the few to tens of copies, in isolated cellular compartments such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number, but lack information on the variance, or they are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling of the cellular compartment with fluorescent primary-secondary antibody complexes, total internal reflection fluorescence microscopic imaging of the cellular compartment, digital image analysis and deconvolution of the fluorescence intensity data.

Cotrafficking of SV2 and synaptotagmin at the synapse.

Synaptic vesicles are specialized cycling endosomes that contain a unique constellation of membrane proteins. Proteins are sorted to vesicles by short amino acid sequences that serve as binding sites for clathrin adaptor proteins. Here we show that a tyrosine-based endocytosis motif in the vesicle protein SV2 is required for trafficking to synaptic vesicles of both SV2 and the calcium sensor protein synaptotagmin.

Large structural change in isolated synaptic vesicles upon loading with neurotransmitter.

The size of a synaptic vesicle (SV) is generally thought to be determined by the amount of lipid and membrane protein it contains. Once formed, it is thought to remain constant in size. Using fluorescence correlation spectroscopy and cryogenic electron microscopy, we show that glutamatergic vesicles reversibly increase their size upon filling with glutamate.