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 the molecular basis of nucleo-cytoplasmic
transport.
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 have also been 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.
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. Planar polarity adds a
further organizational level to tissues.
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. Each of these processes represents a form
of global or local polarization.
Loss of polarity is important in metastasis,
when epithelial cells lose polarity and invade the basement
membrane – a process referred to as the epithelial-mesenchymal
transition (EMT).
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 have been particularly interested
in Cdc42, a small GTPase, in the
PAR genes, and in Septins. 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)
have 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 are also using siRNA
screens to identify new components of the signaling pathways
that control cell polarity in epithelial cells and neurons.
Huaye has also 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 interested
in other polarity proteins, including Scribble,
which in MDCK cells regulates the activity of E-cadherin
(Qin
et al, J Cell Biol, 2005). 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 generate multiple splice variants, and
they 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. Knockout mice have not
been very informative in identifying biological functions
so far, perhaps because several of the septins can play
redundant roles. Silencing by RNAi in cells grown in culture
has, however, revealed a number of interesting roles.
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 important and unexpected
discovery – he found 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 essential
function of all eukaryotic cells (Macara,
MMBR, 2001). Protein cargo destined for the nucleus
is tagged with a nuclear localization signal (NLS) recognized
by specific transport receptors (called karyopherins or
importins). Importins carry their cargo through nuclear
pore complexes, which are large structures plugged through
the double membrane of the nuclear envelope. Within the
nucleus, RanGTP binds to the importin, triggering release
of its cargo.
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).. The unloaded importin,
in complex with RanGTP, recycles back to the nucleus where
GTP hydrolysis is catalyzed by a GTPase-activating protein,
RanGAP. This hydrolysis releases RanGDP, which can be returned
to the nucleus by NTF2. An important cofactor is Npap60
(also called Nup50), which facilitates importin-alpha mediated
nuclear import (Lindsay
et al, Cell, 2002).
Most interestingly, a graduate student in the lab, Ting
Chen, recently 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 are currently purifying the
methyltransferase, and have identified several new substrates
for the enzyme. We believe that this modification is quite
widespread and will have important functions in the cell.
