Secretory vesicle formation and dynamics


The intracellular vesicle transport

The structural and metabolic stability of a eukaryotic cell depends on the trafficking of material between sub-cellular compartments.  Intracellular trafficking requires the formation and correct targeting of small, membrane-bound vesicles.  Vesicles are first created from a so-called donor compartment.  Once fully formed, the vesicles are then actively transported to, and subsequently fuse with, an acceptor compartment where they deliver their soluble cargo into the lumen of the acceptor compartment and release their membrane proteins into the acceptor compartment membrane.  A range of human viral and bacterial pathogens co-opt the vesicle formation machinery to gain entry to their target cells.  In addition, a variety of hereditary diseases are associated with defects in vesicle trafficking, including familial hypercholesterolemia, hereditary hemochromatosis, some types of leukemia, inclusion-cell disease, Griscelli’s syndrome, Hermansky-Pudlak syndrome, arthrogryposis multiplex congenital, renal dysfunction, and cholestasis (ARC) syndrome.  



The formation and transport of vesicles requires protein coats

The formation of a transport vesicle (often called ‘budding’) is not a spontaneous process and requires an input of energy.  Eukaryotic cells have evolved a variety of protein machineries, known as coats, that are designed for this purpose.  In essence, vesicle coats polymerize onto the membrane surface of a donor compartment, simultaneously inducing the collection of cargo molecules into the forming vesicle and providing at least part of the driving force to deform a segment of the donor membrane into a vesicle.  Several different coating systems have been identified, each with unique architectural and dynamic properties.  The distinct structural and mechanistic properties of the different coating systems derives from their use in specific steps the cellular vesicle trafficking network.  For example, the clathrin coating system seems to be primarily employed for a branch of the endocytic pathway (where material from the outside of the cell is internalized) and late stages of the exocytic pathways (where material is transported from the inside of the cell toward the exterior).  By contrast, the COPI system is utilized at the Golgi apparatus to form retrograde moving transport vesicles, and the COPII system at the endoplasmic reticulum to form anterograde moving vesicles.


Fission and fusion

Coat polymerization alone is not sufficient to complete the vesicle budding process.  The collection of membrane through coat assembly inevitably ends with the formation of a narrow membrane neck connecting the budding vesicle to the parent membrane.  Scission of this membrane neck is then required for release of the transport vesicle.  The membrane severing process is generally referred to as ‘fission’ and requires a family of highly specialized protein machines that recognize, wrap and then cut the membrane neck, using the free energy of GTP or ATP hydrolysis to drive the clipping reaction.  Dynamin is the founding member of this large and essential family of membrane severing machines.



Once released from its donor compartment, the transport vesicle must be correctly targeted to its acceptor compartment and the coat must be removed in order for the subsequent delivery of the vesicle’s cargo to take place.  The ultimate fate of a transport vesicle is to merge with its acceptor compartment in a process termed ‘fusion.‘  Importantly, the targeting and fusion of transport vesicles is highly selective.  For example, secretory vesicles derived from the trans-Golgi apparatus carrying material intended for deposition outside the cell are designed to recognize and to fuse only with the plasma membrane, not with an endosome or a lysosome.  This high-fidelity targeting and fusion process involves a complex and elaborated system of protein machines that execute specific tethering, fusion and proof-reading events.  At the core of the regulated fusion machinery are the SNARE proteins, which are anchored in both vesicle and acceptor membranes, and a hydrophilic Sec1/Munc-18 (SM) protein.


Questions

Our lab is interested in a number of unresolved issues that concern how transport vesicles are formed and consumed in a living cell.  While it is known that removal of a vesicle coat must occur prior to membrane fusion, in many cases, exactly when and how vesicle coats are removed is not well understood.  A detailed mechanistic understanding of coat disassembly thus remains elusive.  In addition, membrane fission is essentially the reverse of membrane fusion.  While the membrane intermediates in the fission and fusion reactions are topologically similar, the proteins required for the two jobs are very different.  Membrane fission involves proteins that bind and bend the membrane, where in membrane fusion proteins pierce and pull the membranes together.  Important mysteries remain about how dynamin-like molecules and their accessory proteins drive membrane fission one the one hand, and how SNARE and SM proteins facilitate membrane fusion on the other.   We believe the answer to the question, “How do proteins bring membranes together and pull them apart?” lies in the different mechanical solutions nature has provided to these two similar but distinct events.

One of the ways in which we will approach these questions is by using single particle burst methods like Burst Analysis Spectroscopy (BAS) to probe the complex population dynamics of membrane fusion and fission.  BAS offers a unique tool to observe changes in the size and number of vesicles during fission and fusion, which we will use to test the role of the core fission and fusion machinery, as well as the molecules that regulate them.  With this approach, we hope to provide an answer to the question, “How do cells control membrane fusion and fission?”  

Department of Biochemistry and Biophysics                           Texas A&M University              2017