Research Program
Cell Surface Interactions

Project #
CSI14

Participating Faculty:	F. Maxfield and B. Baird
NBTC Students/Postdocs:	Ethan Chiang and Inna Grosheva
Other Students/Postdocs:	Dana Cruz

Objectives
Macrophages are immune system cells that carry out a variety of functions throughout the body.  One function of these cells is to clear away extracellular debris such as dead or dying cells and denatured proteins.  In some cases, the material that needs to be digested is as large or larger than the macrophages themselves.  Microglia carry out a similar function in the central nervous system.  We will test the hypothesis that these cells create an external, acidified, sealed compartment and that they secrete digestive enzymes into this compartment to create an external “lysosomal synapse”.  We will use patterned surfaces containing either lipoproteins or Alzheimer’s amyloid fibrils and examine the interaction of macrophages and microglia with these surfaces.

Methods
Micro- and nanofabrication including photolithography for parylene lift-off or ZMWs; patterned lipid bilayers and proteins; electron microscopic tomography of cellular structures; fluorescence confocal microscopy; total internal reflection fluorescence microscopy.

Summary
One biologically and medically important function of macrophages is their interaction with aggregated lipoproteins in the wall of blood vessels leading to massive uptake of cholesterol and conversion of Mfs to foam cells, which is an early step in the development of atherosclerosis.  Mfs encounter these extracellular matrix-linked aggregated lipoprotein particles, which are as large as a few hundred nm, in arterial blood vessels in regions of high turbulence.  This material cannot be taken up by conventional endocytosis because of its size and its linkage to the matrix polymers; nevertheless, the cholesteryl esters in the core of these agLDL particles are efficiently hydrolyzed by lysosomal acid lipase, delivering a large load of cholesterol to the cells. We have recently found evidence for a novel mechanism in which cells form an extracellular acidified lysosomal compartment (the lysosomal synapse).  This compartment appears to be similar to a compartment formed between bone and osteoclasts.      

The micropatterned surfaces provide a unique opportunity to demonstrate the properties of a novel cell-associated structure - the lysosomal synapse.  Having well defined patterned structures of proteins that are tightly anchored to the glass substrate will allow us to be certain that the structures we are studying are extracellular.  The ability to adjust the size of the spots of proteins will allow us to mimic the size of objects encountered in vivo and evaluate possible size dependence.  Combining this with TIRF imaging and tomographic electron microscopy will allow us to examine these structures and their properties at both microscale and nanoscale resolution.  We have good preliminary data suggesting that hydrolysis of lipoproteins occurs in the lysosomal synapse in a model system that mimics early events in atherosclerotic lesions.  This opens the possibility that inhibition of cholesteryl ester hydrolysis in this extracellular compartment could be used to slow the cholesterol loading of Mfs, thus slowing foam cell formation. 

If we can demonstrate that similar extracellular hydrolysis of deposits of fAb take place in lysosomal synapses of microglia, activation of this process could lead to new ways to clear amyloid plaques from the brains of Alzheimer’s disease patients.

Ultrastructural studies of the lysosomal synapse by electron microscopic tomography will be very useful for visualizing the cellular structures at the contacts necessary to form a compartment that is sealed well enough to maintain a pH gradient.