Our long-term goal is to understand the mechanisms by which epithelial cells develop structural and functional asymmetry. This process is critical to the normal function of epithelial tissues and loss of epithelial polarity is associated with a varity of human pathologies including cancers, cystic diseases, secretory and absorptive disorders and cardiovascular disease. An evolutionarily conserved event in cell polarization involves cortical capture and selective stabilization of microtubules (MTs). In stabilizing a subset of MTs selectively, cells develop an axis of morphological asymmetry along which local signals are transduced and on which MT motor proteins carry diverse cargoes to and from specific domains inside and on surface of cells. While it is known that signaling from specific cortical sites triggrs the local MT reorganization necessary for polarization, the molecules and regulatory mechanisms orchestrating these processes have not been elucidated fully. We have found that the kinesin-2 family motor, KIF17, is a key modulator of MT capture and stabilization and that contributes to polarization of epithelial cells. Based on analogy with polarization mechanisms in single-cell organisms, we hypothesize this kinesin acts as a selective transporter of factors regulating MT stabilization and cell polarity in epithelia. However, the relevant cargoes transported by this motor that could promote MT stabilization and cell polarity are still unknown. Furthermore, the mechanisms that control KIF17 activity in cells have not been explored. We will use a combination of approaches to identify epithelial cargoes of KIF17 and test if they too regulate epithelial polarization. In addition, we will characterize the mechanism by which this kinesin and its cargoes regulate cell polarity using a combination of biochemical and advanced imaging approaches to monitor activity of this motor directly in vitro and in living cells. Another important process in epithelial polarization involves the selective and targeted transport of membrane and secreted proteins to different regions of the cell surface. However, little is known about how cellular cargoes select appropriate motor(s) to get them to the correct destination and how motors identify appropriate routes to these destinations. We showed recently the existence of polarization-dependent, "kinesin-switching" by an apical membrane protein cargo being transported from the Golgi to the plasma membrane. This leads us to hypothesize that kinesin interactions with specific cargoes are regulated during epithelial polarization. This kinesin-cargo switch likely facilitates development of functional membrane asymmetry in response to MT reorganization. To test idea we will identify proteins that regulate the binding of kinesin to vesicles containing the apical membrane protein p75. We will then characterize how kinesin-vesicle interactions are regulated during epithelial polarization. We will expand this aim by applying new molecular tools developed in the lab that allow us to monitor kinesin activation directly when cargo is presented in living cells. Understanding the role of kinesins in epithelial polarization may provide critical new information about how this process is controlled and could lead to identification of novel therapeutic targets in treating diseases of epithelial origin.

Public Health Relevance

Polarized epithelial cells play a key role in maintaining internal homeostasis by acting as a barrier between the outside environment and internal organs. Our long-term goal is to understand, at molecular and mechanistic levels, how epithelial cells develop structural and functional symmetry. This process is critical to the normal function of tissues comprised of these cells. Indeed, loss of epithelial polarity is associated with a variety f human pathologies including cancers of diverse origin, cystic disease and malabsorption and secretory disorders in several organ systems. Elucidation of molecular events driving polarization is important, not only for understanding normal development, but also because the details of this process will enable us to develop therapeutic intervention strategies targeting disease associated with loss of epithelial polarity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM087575-04S1
Application #
8573173
Study Section
Special Emphasis Panel (ZGM1-CBB-0 (MI))
Program Officer
Gindhart, Joseph G
Project Start
2009-04-01
Project End
2014-12-31
Budget Start
2013-09-01
Budget End
2013-12-31
Support Year
4
Fiscal Year
2013
Total Cost
$56,499
Indirect Cost
$23,166
Name
Weill Medical College of Cornell University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Acharya, Bipul R; Espenel, Cedric; Kreitzer, Geri (2013) Direct regulation of microtubule dynamics by KIF17 motor and tail domains. J Biol Chem 288:32302-13
Espenel, Cedric; Acharya, Bipul R; Kreitzer, Geri (2013) A biosensor of local kinesin activity reveals roles of PKC and EB1 in KIF17 activation. J Cell Biol 203:445-55
Xing, Bowen; Wang, Limin; Guo, Dagang et al. (2013) Atypical protein kinase Cýý is critical for growth factor receptor-induced dorsal ruffle turnover and cell migration. J Biol Chem 288:32827-36
Nekrasova, Oxana E; Amargo, Evangeline V; Smith, William O et al. (2011) Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes. J Cell Biol 195:1185-203
Jaulin, Fanny; Kreitzer, Geri (2010) KIF17 stabilizes microtubules and contributes to epithelial morphogenesis by acting at MT plus ends with EB1 and APC. J Cell Biol 190:443-60