Quantifying Intramolecular Protein Conformational Dynamics Under Lipid Interaction Using smFRET and FCCS.

FÓ§rster-type resonance energy transfer (FRET) with fluorescence cross-correlation spectroscopy (FCCS) is a powerful combination for observing intramolecular conformational dynamics on the micro- to millisecond timescale. Owing to its sensitivity to various physical parameters, FRET-FCCS has also been used to detect the reagent effects on proteins dynamics. However, FRET-FCCS alone cannot acquire the exact measurements of rate constants. Moreover, this technique is highly model dependent and can be unreliable when determining too many parameters at once. On the contrary, single-molecular FRET (smFRET) can measure the conformational states and their populations directly, although it is extremely challenging for probing fast dynamics under 1 ms. In this chapter, we describe how to realize sub-millisecond conformational dynamics measurements of a SNARE protein Ykt6 under lipid environments by smFRET and FRET-FCCS. This protocol includes sample preparation, microscope designs, data acquisition, and analysis methodology.

Human Platelet Vesicles Exhibit Distinct Size and Proteome.

In the past 50 years, isolated blood platelets have had restricted use in wound healing, cancer therapy, and organ and tissue transplant, to name a few. The major obstacle for its unrestricted use has been, among others, the presence of ultrahigh concentrations of growth factors and the presence of both pro-angiogenic and anti-angiogenic proteins. To overcome this problem requires the isolation and separation of the membrane bound secretory vesicles containing the different factors. In the current study, high-resolution imaging of isolated secretory vesicles from human platelets using atomic force microscopy (AFM) and mass spectrometry enabled characterization of the remaining vesicles size and composition following their immunoseparation. The remaining vesicles obtained following osmotic lysis, when subjected to immunoseparation employing antibody to different vesicle-associated membrane proteins (VAMPs), demonstrate for the first time that VAMP-3-, VAMP-7-, and VAMP-8-specific vesicles each possesses distinct size range and composition. These results provide a window into our understanding of the heterogeneous population of vesicles in human platelets and their stability following both physical manipulation using AFM and osmotic lysis of the platelet. This study further provides a platform for isolation and the detailed characterization of platelet granules, with promise for their future use in therapy. Additionally, results from the study demonstrate that secretory vesicles of different size found in cells reflect their unique and specialized composition and function.

Super-resolution imaging prompts re-thinking of cell biology mechanisms: selected cases using stimulated emission depletion microscopy.

The use of super-resolution imaging techniques in cell biology has yielded a wealth of information regarding cellular elements and processes that were invisible to conventional imaging. Focusing on images obtained by stimulated emission depletion (STED) microscopy, we discuss how the new high-resolution data influence the ways in which we use and interpret images in cell biology. Super-resolution images have lent support to some of our current hypotheses. But, more significantly, they have revealed unexpectedly complex processes that cannot be accounted for by the simpler models based on diffraction-limited imaging. The super-resolution imaging data challenge cell biologists to change their theoretical framework, by including, for instance, interpretations that describe multiple functions, functional errors or lack of function for cellular elements. In this context, we argue that descriptive research using super-resolution microscopy is now as necessary as hypothesis-driven research.

Unaltered SNARE complex formation in an in vivo model of prion disease.

The ME7 model of prion disease is a chronic slowly evolving model of neurodegeneration in which cell death is preceded by synaptic dysfunction. Previous studies in cell culture show that accumulation of misfolded prion inhibits the formation of the SNARE complexes involving synaptobrevin, syntaxin and SNAP-25 that play an essential role in neurotransmitter release. Such observations suggest that similar phenomenon may contribute to synaptic dysfunction observed in vivo. We have thus used detergent extraction of hippocampal tissue to investigate the status of SNARE complexes in the ME7 model. In the presence of increasing PrP(Sc) deposition we failed to see a change in the amount of SNARE complexes directly extracted into SDS and resolved by SDS-PAGE. Conversely pre-extraction in Triton X-100, a treatment that promotes SNARE complexes ex vivo, demonstrated a modest reduction in hippocampal SNARE complexes when homogenates were made from tissue at late stage disease. This suggests that accumulated PrP(Sc), or perhaps fibrillar complexes formed of prion only inhibit SNARE complexes that are formed ex vivo following biochemical extraction. Thus the accumulation of PrP(Sc) although deleterious to synaptic function in vivo, does not exert its synaptic effects by disrupting the formation of SNARE complexes that are core to transmitter release.

Selective membrane protein internalization accompanies movement from the endoplasmic reticulum to the protein storage vacuole pathway in Arabidopsis.

In plant cells, certain membrane proteins move by unknown mechanisms directly from the endoplasmic reticulum (ER) to prevacuolar or vacuole-like organelles where membrane is internalized to form a dense, lattice-like structure. Here, we identify a sequence motif, PIEPPPHH, in the cytoplasmic tail of a membrane protein that directs the protein from the ER to vacuoles where it is internalized. A type II membrane protein in Arabidopsis thaliana, (At)SRC2 (for Soybean Gene Regulated by Cold-2), binds specifically to PIEPPPHH and moves from the ER to the same vacuoles where it is internalized. Not all proteins that move in this pathway are internalized because another Arabidopsis type II membrane protein, (At)VAP (for Vesicle-Associated Protein), localizes to the same organelles but remains exposed on the limiting membrane. The identification of (At)SRC2 and its preference for interaction with a targeting motif specific for the ER-to-vacuole pathway may provide tools for future dissection of mechanisms involved in this unique trafficking system.