Research in the Sheff Lab
A summary of
our Research:
The mechanisms
by which cells generate and maintain polarity are among the most fundamental in
all of biology. Especially intriguing,
and pharmacologically important, is the question of how cells maintain the
different compositions of their apical and basolateral surfaces in the face of
rapid and continuous internalization of the plasma membrane. My research is focused on how proteins
internalized from either surface navigate the endosomal system to return to the
cell surface. To approach these
problems, I use a combination of cutting edge digital microscopy, proteomic
analysis and biochemical assays.
Most drugs, hormones, chemical
transmitters and other ligands operate by binding to receptors on the plasma
membrane of target cells.
Receptors are continuously internalized and delivered to early
endosomes. To maintain receptor
levels at the cell surface, the receptors are recycled to the plasma
membrane. Receptors recycle either
directly to the plasma membrane or through recycling endosomes. This second pathway is important
because receptors and membrane are stored in recycling endosomes.
Polarized cells such as epithelial cells,
neurons, or hepatocytes are more complex.
The plasma membranes of these cells have apical and basolateral domains
with different protein and lipid compositions. Receptors from each domain are internalized to separate
populations of early endosomes, but traffic from both surfaces meets in a
single set of recycling endosomes on the apical side of the nucleus. Surprisingly, receptors from this
common compartment are sorted and delivered back to the correct plasma membrane
domain. A receptor may pass
through recycling endosomes over 100 times during its lifetime, so that even
minimal mis-sorting as occurs in familial hypercholesterolemia, familial
hypertension and some forms of cystic fibrosis, causes life-threatening
disease.
The process by
which recycling endosomes sort proteins for apical or basolateral delivery is
little understood. While the
signals governing sorting are known, the mechanism for 
interpreting those signals is not. We have recently observed that apical
and basolateral receptors segregate within recycling endosomes to form distinct
apical and basolateral subdomains.
Other proteins, such as Rab11 and Rab8 GTPases colocalize with the
apical and basolateral subdomains suggesting that they may play a role in
sorting of the receptors. These
findings allow us for the first time to correlate morphological data within the
recycling endosomes with quantitative kinetic data derived from the effects of
mutant Rab proteins on receptor recycling traffic. As a result we can begin dissect what role each protein may
play in sorting of apical from basolateral traffic.
While the aim of
this research is a fundamental understanding of basic cellular processes, many
of the proteins involved in recycling endosome traffic regulation may
themselves be useful targets for new therapeutic drugs. Such drugs would change levels of key
receptors by altering traffic at the endosomal level or by retargeting
receptors to the opposite plasma membrane domain.
Selected
Publications:
Pelletier, L.
Stern, C. A. Pypaert, M. Sheff, D.
Ngo, H. M. Roper, N. He, C. Y. Hu, K. Toomre, D. Coppens, I. Roos, D. S. Joiner,
K. A. and G. Warren. 2002. Golgi biogenesis in Toxoplasma gondii Nature 418:548-552
David R. Sheff, L. Pelletier, C. O'Connell, G. Warren,
and I. Mellman. 2002.
Transferrin receptor recycling in the absence of perinuclear recycling endosomes. J. Cell Biol. 156: 797-804
David
R. Sheff, R. Krochewski
and I. Mellman. 2002. Actin
dependence of polarized receptor recycling in MDCK cell endosomes. Mol.
Biol. Cell. 13:262-275
David R.
Sheff, E. A. Daro and I.
Mellman. 1999. The endocytic recycling pathway contains two distinct
populations of early endosomes with different sorting functions. J. Cell Biol. 145:123-139.