Imaging is used to map activity across populations of neurons. Microscopes with cellular resolution have small (< 1 millimeter) fields of view and cannot simultaneously image activity distributed across multiple brain areas. Typical large field of view microscopes do not resolve single cells, especially in the axial dimension. We developed a 2-photon random access mesoscope (2p-RAM) that allows high-resolution imaging anywhere within a volume spanning multiple brain areas (Φ 5 mm x 1 mm cylinder). 2p-RAM resolution is near diffraction limited (lateral, 0.66 μm, axial 4.09 μm at the center; excitation wavelength = 970 nm; numerical aperture = 0.6) over a large range of excitation wavelengths. A fast three-dimensional scanning system allows efficient sampling of neural activity in arbitrary regions of interest across the entire imaging volume. We illustrate the use of the 2p-RAM by imaging neural activity in multiple, non-contiguous brain areas in transgenic mice expressing protein calcium sensors.
Animal experimentation: All procedures were in accordance with protocols approved by the Janelia Research Campus Institutional Animal Care and Use Committee. IACUC 14-115.
- Fred Rieke, Howard Hughes Medical Institute, University of Washington, United States
© 2016, Sofroniew et al.
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Exocytosis of secretory vesicles requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and small GTPase Rabs. As a Rab3/Rab27 effector protein on secretory vesicles, Rabphilin 3A was implicated to interact with SNAP-25 to regulate vesicle exocytosis in neurons and neuroendocrine cells, yet the underlying mechanism remains unclear. In this study, we have characterized the physiologically relevant binding sites between Rabphilin 3A and SNAP-25. We found that an intramolecular interplay between the N-terminal Rab-binding domain and C-terminal C2AB domain enables Rabphilin 3A to strongly bind the SNAP-25 N-peptide region via its C2B bottom α-helix. Disruption of this interaction significantly impaired docking and fusion of vesicles with the plasma membrane in rat PC12 cells. In addition, we found that this interaction allows Rabphilin 3A to accelerate SNARE complex assembly. Furthermore, we revealed that this interaction accelerates SNARE complex assembly via inducing a conformational switch from random coils to α-helical structure in the SNAP-25 SNARE motif. Altogether, our data suggest that the promotion of SNARE complex assembly by binding the C2B bottom α-helix of Rabphilin 3A to the N-peptide of SNAP-25 underlies a pre-fusion function of Rabphilin 3A in vesicle exocytosis.
3′ end formation of most eukaryotic mRNAs is dependent on the assembly of a ~1.5 MDa multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 3′ end processing machinery onto which the remaining factors, including cleavage stimulation factor (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal ‘acidic’ region of hFip1. In turn, CstF77 competitively inhibits CPSF-dependent PAP recruitment and 3′ polyadenylation. Taken together, these results provide a structural basis for the multivalent scaffolding and regulatory functions of hFip1 in 3′ end processing.