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Membrane-directed molecular assembly of the neuronal SNARE complex.

Cho WJ, Lee JS, Zhang L, Ren G, Shin L, Manke CW, Potoff J, Kotaria N, Zhvania MG, Jena BP - J. Cell. Mol. Med. (2011)

Bottom Line: Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution.Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study.Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membrane-directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.

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Schematic flow diagram establishing membrane-associated SNARE complex assembly and its structural evaluation using AFM and EM. (A) Preteoliposomes of a specific size containing v-SNARE can be exposed to t-SNARE-reconstituted lipid membrane supported by a mica surface. The interaction results in the formation of a highly stable t-/v-SNARE ring complex which can be observed using AFM following removal of the vesicle. (B) Similarly, proteoliposomes of a certain size, one reconstituted with v-SNARE and the other with t-SNARE, can interact (C, D) to form membrane-directed t-/v-SNARE complex. However, in this situation, a single t-SNARE vesicle (red) is physically limited to establish contact with a maximum of 12 (blue) equal-size v-SNARE vesicles, and vice versa. This allows for approximately no more than 6% of the total surface area of one vesicle to interact with others, translating the membrane-directed t-/v-SNARE complex to be just around 6% of the total. In order to observe this membrane-directed SNARE complex, the liposomes are solubilized using detergent, and the stable t-/v-SNARE ring complex formed as a result of membrane-directed assembly, is then imaged using AFM and EM. (C) Schematic diagram depicting the possible molecular mechanism of SNARE ring complex formation, when t-SNARE-vesicles and V-SNARE-vesicles meet. The process may occur due to a progressive recruitment of t-/v-SNARE pairs as the opposing vesicles are pulled toward each other, until a complete ring is established, preventing any further recruitment of t-/v-SNARE pairs to the complex. The top panel is a side view of two vesicles (one t-SNARE-reconstituted, and the other v-SNARE reconstituted) interacting to form a single t-/v-SNARE complex, leading progressively (from left to right) to the formation of the ring complex. The lower panel is a top view of the two interacting vesicles.
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fig01: Schematic flow diagram establishing membrane-associated SNARE complex assembly and its structural evaluation using AFM and EM. (A) Preteoliposomes of a specific size containing v-SNARE can be exposed to t-SNARE-reconstituted lipid membrane supported by a mica surface. The interaction results in the formation of a highly stable t-/v-SNARE ring complex which can be observed using AFM following removal of the vesicle. (B) Similarly, proteoliposomes of a certain size, one reconstituted with v-SNARE and the other with t-SNARE, can interact (C, D) to form membrane-directed t-/v-SNARE complex. However, in this situation, a single t-SNARE vesicle (red) is physically limited to establish contact with a maximum of 12 (blue) equal-size v-SNARE vesicles, and vice versa. This allows for approximately no more than 6% of the total surface area of one vesicle to interact with others, translating the membrane-directed t-/v-SNARE complex to be just around 6% of the total. In order to observe this membrane-directed SNARE complex, the liposomes are solubilized using detergent, and the stable t-/v-SNARE ring complex formed as a result of membrane-directed assembly, is then imaged using AFM and EM. (C) Schematic diagram depicting the possible molecular mechanism of SNARE ring complex formation, when t-SNARE-vesicles and V-SNARE-vesicles meet. The process may occur due to a progressive recruitment of t-/v-SNARE pairs as the opposing vesicles are pulled toward each other, until a complete ring is established, preventing any further recruitment of t-/v-SNARE pairs to the complex. The top panel is a side view of two vesicles (one t-SNARE-reconstituted, and the other v-SNARE reconstituted) interacting to form a single t-/v-SNARE complex, leading progressively (from left to right) to the formation of the ring complex. The lower panel is a top view of the two interacting vesicles.

Mentions: Because majority of intracellular fusion events occur between vesicular compartments, it was imperative to determine the structure and arrangement of the t-/v-SNARE complex when t-SNARE-vesicles and v-SNARE-vesicles meet. In this case, because both opposing membrane would exhibit curvature, we expected the v- and t-SNAREs to interact at a point, attracting additional SNARE pairs to form a t-/v-SNARE bundle. To test our hypothesis, two sets of 50 nm diameter liposomes were reconstituted with t-SNAREs and v-SNARE respectively (Fig. 1A–C), for use in our study. Surprisingly, exposure of 50 nm v-SNARE-liposome to 50 nm t-SNARE-liposome resulted in the establishment of a highly stable approximately 8 nm diameter t-/v-SNARE ring complex. Once formed, the t-/v-SNARE complex is highly stable, resistant even to a membrane solubilizing anionic detergent, sodium dodecyl sulphate. Therefore, following membrane solubilization (Fig. 1B, D), the stable SNARE complexes [8] have enabled a detailed examination of its morphology at high resolution, using both AFM and electron microscopy (EM) in the current study.


Membrane-directed molecular assembly of the neuronal SNARE complex.

Cho WJ, Lee JS, Zhang L, Ren G, Shin L, Manke CW, Potoff J, Kotaria N, Zhvania MG, Jena BP - J. Cell. Mol. Med. (2011)

Schematic flow diagram establishing membrane-associated SNARE complex assembly and its structural evaluation using AFM and EM. (A) Preteoliposomes of a specific size containing v-SNARE can be exposed to t-SNARE-reconstituted lipid membrane supported by a mica surface. The interaction results in the formation of a highly stable t-/v-SNARE ring complex which can be observed using AFM following removal of the vesicle. (B) Similarly, proteoliposomes of a certain size, one reconstituted with v-SNARE and the other with t-SNARE, can interact (C, D) to form membrane-directed t-/v-SNARE complex. However, in this situation, a single t-SNARE vesicle (red) is physically limited to establish contact with a maximum of 12 (blue) equal-size v-SNARE vesicles, and vice versa. This allows for approximately no more than 6% of the total surface area of one vesicle to interact with others, translating the membrane-directed t-/v-SNARE complex to be just around 6% of the total. In order to observe this membrane-directed SNARE complex, the liposomes are solubilized using detergent, and the stable t-/v-SNARE ring complex formed as a result of membrane-directed assembly, is then imaged using AFM and EM. (C) Schematic diagram depicting the possible molecular mechanism of SNARE ring complex formation, when t-SNARE-vesicles and V-SNARE-vesicles meet. The process may occur due to a progressive recruitment of t-/v-SNARE pairs as the opposing vesicles are pulled toward each other, until a complete ring is established, preventing any further recruitment of t-/v-SNARE pairs to the complex. The top panel is a side view of two vesicles (one t-SNARE-reconstituted, and the other v-SNARE reconstituted) interacting to form a single t-/v-SNARE complex, leading progressively (from left to right) to the formation of the ring complex. The lower panel is a top view of the two interacting vesicles.
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Related In: Results  -  Collection

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fig01: Schematic flow diagram establishing membrane-associated SNARE complex assembly and its structural evaluation using AFM and EM. (A) Preteoliposomes of a specific size containing v-SNARE can be exposed to t-SNARE-reconstituted lipid membrane supported by a mica surface. The interaction results in the formation of a highly stable t-/v-SNARE ring complex which can be observed using AFM following removal of the vesicle. (B) Similarly, proteoliposomes of a certain size, one reconstituted with v-SNARE and the other with t-SNARE, can interact (C, D) to form membrane-directed t-/v-SNARE complex. However, in this situation, a single t-SNARE vesicle (red) is physically limited to establish contact with a maximum of 12 (blue) equal-size v-SNARE vesicles, and vice versa. This allows for approximately no more than 6% of the total surface area of one vesicle to interact with others, translating the membrane-directed t-/v-SNARE complex to be just around 6% of the total. In order to observe this membrane-directed SNARE complex, the liposomes are solubilized using detergent, and the stable t-/v-SNARE ring complex formed as a result of membrane-directed assembly, is then imaged using AFM and EM. (C) Schematic diagram depicting the possible molecular mechanism of SNARE ring complex formation, when t-SNARE-vesicles and V-SNARE-vesicles meet. The process may occur due to a progressive recruitment of t-/v-SNARE pairs as the opposing vesicles are pulled toward each other, until a complete ring is established, preventing any further recruitment of t-/v-SNARE pairs to the complex. The top panel is a side view of two vesicles (one t-SNARE-reconstituted, and the other v-SNARE reconstituted) interacting to form a single t-/v-SNARE complex, leading progressively (from left to right) to the formation of the ring complex. The lower panel is a top view of the two interacting vesicles.
Mentions: Because majority of intracellular fusion events occur between vesicular compartments, it was imperative to determine the structure and arrangement of the t-/v-SNARE complex when t-SNARE-vesicles and v-SNARE-vesicles meet. In this case, because both opposing membrane would exhibit curvature, we expected the v- and t-SNAREs to interact at a point, attracting additional SNARE pairs to form a t-/v-SNARE bundle. To test our hypothesis, two sets of 50 nm diameter liposomes were reconstituted with t-SNAREs and v-SNARE respectively (Fig. 1A–C), for use in our study. Surprisingly, exposure of 50 nm v-SNARE-liposome to 50 nm t-SNARE-liposome resulted in the establishment of a highly stable approximately 8 nm diameter t-/v-SNARE ring complex. Once formed, the t-/v-SNARE complex is highly stable, resistant even to a membrane solubilizing anionic detergent, sodium dodecyl sulphate. Therefore, following membrane solubilization (Fig. 1B, D), the stable SNARE complexes [8] have enabled a detailed examination of its morphology at high resolution, using both AFM and electron microscopy (EM) in the current study.

Bottom Line: Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution.Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study.Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membrane-directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.

Show MeSH
Related in: MedlinePlus