Cell Polarity and Nuclear Transport in Epithelia, Neurons, and Cancer
Background:
Our lab is interested in the ways in which cells break symmetry, and initiate and maintain spatial asymmetries. Asymmetries can form as cells polarize, or can occur within cells, for instance during mitosis. Gradients of proteins or other molecules can create asymmetries that the cell uses to specify orientations and subcellular locations. Cell polarity is usually lost early during tumor progression, and several polarity proteins have been identified as tumor suppressors.
We have 4 major programs ongoing – one on the mechanisms that determine the polarization of mammalian epithelial cells and hippocampal neurons; a related program on the role of polarity proteins in mammary gland morphogenesis and in the initiation of breast cancer; one on the role of septins in cell polarity; and one on a novel post-translational modification of proteins, called α-N-methylation. Related to our studies on cell polarity, we are also interested in the mechanisms that control RNA localization.
Approaches:
We use a range of cell biological approaches to study these cell processes, including micro-injection and transfection, live-cell 4D imaging and immunofluorescence, FRET biosensors, permeabilized cell assays, two-hybrid screens, siRNA screens, and other molecular biological technologies. We are also developing technologies to rapidly assess gene function in organogenesis, using mammary transplants and organoid cultures of stem cells in which gene expression has been manipulated using lentivirus. We recently performed a genome-wide screen to identify RNAs that localize to protrusions at the leading edge of migrating cells.
Cell polarity:
A fundamental biological question is how cells establish and maintain a polarized morphology (Macara, Nature Mol. Biol. Reviews, 2003Macara, Nature Mol. Biol. Reviews, 2003; Goldstein & Macara, Dev Cell 2007). Some form of polarization is common to almost all cells in all organisms, even among the Prokarya, but is particularly sophisticated in higher organisms, in which cells organize into tissues. Epithelial sheets are the fundamental building blocks of the metazoa, and were probably the earliest tissue type to evolve.
The formation of epithelial sheets depends on two properties – apical/basal polarization and adhesion. Adhesion between cells produces the sheet; and polarization determines the orientation of the sheet. The elaboration of this simple idea has led to the complex tubular organization of many major tissues and organs.
Other important examples of polarization occur in the elaboration of the nervous system. During neuronal development, a single axon must be specified, while other extensions become dendrites. The dendrites branch and form synapses through thousands of small spines, which extend from the dendritic shaft. Dendritic spines are the main pathway for excitatory input in neurons and are essential for cognition.
Loss of polarity is also of key important in metastasis, when epithelial cells invade the basement membrane – a process referred to as the epithelial-mesenchymal transition (EMT). Over 90% of solid tumors arise from epithelial cells, and early dissemination of transformed cells to distant sites is the leading cause of death from cancer. We believe that many polarity proteins act as tumor suppressors, by preventing dissemination.
A pivotal question is whether all of these different types of cell polarity involve the same genes.
Polarity proteins:
Genetic screens in flies, worms, and yeast have uncovered a large number of conserved genes that are required for normal cell polarity. We are particularly interested in the PAR genes. PAR-6 contains a PDZ domain, which mediates protein-protein interactions. We found that PAR-6 proteins interact with Cdc42-GTP, with another polarity protein, PAR-3, which contains 3 PDZ domains, and with atypical protein kinase C (Joberty et al. Nature Cell Biol. 2000). Lin Gao, a former graduate student, found that PAR-6 can inhibit the assembly of tight junctions in epithelial cells (Gao et al. Current Biol. 2003). In collaboration with Ben Margolis (U. Michigan), PAR-6 was found to interact with another polarity complex through a protein called Pals1 (Hurd et al. Nature Cell Biol. 2003). We obtained crystals of a Cdc42-Par6 complex and in collaboration with Diana Tomchick (UT Southwestern) we solved the structure of this complex to 2.3 Angstroms resolution (Garrard et al, EMBOJ, 2003).
In Drosophila, PAR-3 associates with a protein called Inscuteable, which is essential for the oriented cell divisions of neuroblasts. Insc operates through binding to a protein called Partner of Inscuteable, Pins, which can also bind to certain G-proteins. Quansheng Du, a former postoc in the lab, discovered that mammalian Pins also associates with a microtubule-binding protein called NuMA, and that Pins functions as a molecular switch to control the association of NuMA with the cell cortex (Du & Macara, Cell, 2004).
Xinyu Chen, another former postdoc in the lab, discovered that PAR-3 regulates another small GTPase, Rac, by spatially restricting a Rac exchange factor called Tiam1. (Chen & Macara, Nature Cell Biol., 2005). Interestingly, this signaling pathway is critical not only for the assembly of tight junctions in epithelial cells but also – as was discovered by Huaye Zhang - for dendritic spine morphogenesis in hippocampal neurons (Zhang & Macara, Nature Cell Biol, 2006).
PAR-3 also regulates actin dynamics through cofilin. Xinyu discovered that PAR-3 inhibits LIMK2, which phosphorylates and inactivates cofilin, a small protein that can sever actin filaments (Chen & Macara, J. Cell Biol. 2006).
PAR-3 is known to play an important role in the apical/basal domain organization of epithelial cells in both flies and worms. When PAR-3 expression is silenced in MDCK epithelial cells a defect in lumen formation occurs when the cells are grown in 3D cultures. Instead of forming a single lumen, with apical surface facing inwards, the cells form multiple lumens. Strikingly, this function for PAR-3 in organizing apical/basal polarization is independent of TIAM1 and Rac. Therefore, PAR-3 acts through distinct pathways in different contexts – and these pathways can be switched simply by changing the geometry of the culture conditions.
We are now studying PAR-3 function in the morphogenesis of mammary glands in mice. Dr. Luke McCaffrey in the lab is leading this project. We isolate mammary stem cells from mice, transduce them with lentivirus to manipulate gene expression, then transplant the cells back into a host mouse. The stem cells will regenerate an entire new mammary gland, within a couple of months. We can then determine whether the gene we have manipulated is essential for normal mammary morphogenesis. Silencing of PAR-3, for instance, causes a severe morphogenetic defect (McCaffrey & Macara, Genes & Development, in press).
Huaye recently found a
novel signaling function for PAR-6 in hippocampal neurons, where it
regulates the Rho GTPase through p190RhoGAP (Dev Cell in press).
We are also using siRNA screens to identify new components of the signaling pathways that control cell polarity in epithelial cells and neurons.
We are also interested in other polarity proteins, including Scribble, which in MDCK cells regulates the activity of E-cadherin (Qin et al, J Cell Biol, 2
005). Silencing of Scrb by shRNAs causes a severe defect in cell-cell adhesion, but without any apparent loss of E-cadherin from the cell surface. We are pursuing the underlying molecular mechanism for this regulation.
Septins:
Septins are GTP-binding proteins that can assemble into hetero-oligomers that form filaments. They were discovered in budding yeast, where they form a diffusion barrier between the mother and daughter cells, and are necessary for morphogenetic checkpoints that ensure the cytoskeleton is correctly arranged for mitosis.
Little has been known about the functions of septins in mammalian cells until recently. There are 14 septin genes in humans, which can associate in a variety of heteromic oligomers. In vitro they can also form filaments, but little was understood about how these are organized. Recently, however, the crystal structure of a septin 2/6/7 complex was determined, and this information will be invaluable in decoding the rule that govern septin oligomerization.
A graduate student in the lab, Brandon Kremer, performed a preparative-scale IP of Sept6, and in a proteomics analysis identified the microtubule binding protein, MAP4. He showed that MAP4 binding to microtubules is inhibited by septins, suggesting that one function of septins is to reduce microtubule stability (Kremer et al., MBC 2005).
He also found that depletion of septins by RNAi causes a serious defect in actin organization, and his studies on this defect led to an unexpected discovery – that septins are coupled to the DNA damage response pathway, through the NCK adapter protein. Remarkably, NCK enters the nucleus in response to DNA damage, and is essential for p53 phosphorylation and cell cycle arrest. NCK is carried into the nucleus by SOCS7, which in turn can bind to septins. Through a signaling pathway we do not yet fully understand, DNA damage triggers the dissociation of septins and the rapid accumulation of both SOCS7 and NCK in the nucleus, where NCK regulates p53 phosphorylation. Simultaneously, the loss of NCK from the cytoplasm results in a dramatic remodeling of the actin cytoskeleton (Kremer et al. Cell, 2007). We are following up these findings using both mouse models to study the biological consequences and biochemical approaches to identify further factors in this novel signaling system.
Nucleo-cytoplasmic transport:
One of our research goals is to understand the role of the Ran GTPase and its binding partners in nuclear protein transport. Nuclear transport is an essentia
l function of all eukaryotic cells (Macara, MMBR, 2001).
An important cofactor for nuclear transport is Npap60 (Lindsay et al, Cell, 2002).
The guanine nucleotide exchange factor for Ran (RCC1) is localized to the nucleus, where it generates RanGTP and binds to histones H2A/B (Nemergut et al, Science. 2001). Most interestingly, a graduate student in the lab, Ting Chen, discovered that the exchange factor RCC1 is modified in an unusual way: the N-terminal Met residue is cleaved and the exposed alpha-N-amino group become methylated (Chen et al, Nature Cell Biol, 2007). Alpha-N-methylation has not previously been studied, and no enzymes have been found in animals that catalyze this reaction. Ting identified an enzyme activity in nuclear extracts that drives alpha-N-methylation, and identified the motif that the enzyme recognizes. She also discovered that methylation of RCC1 is essential for the normal association of the protein with chromatin. Mutants that cannot be methylated do not bind efficiently, and disrupt the RanGTP gradient, which leads to mitotic defects and chromosome mis-segregation. We have recently identified this enzyme, and found several important new substrates.