Lecture 4 January 19, 2011
Organizers of intercellular adherens and tight junctions:
nectin and afadin
Most of the information on this section is from the three review articles cited in the notes and slides.
As with other notes, information is often taken verbatim with modification to emphasize
information presented in the lecture.
In the previous classes, you were shown that cell-cell adhesion is strengthened by the
bridging of AJs and TJs to with the actin cytoskeleton and desmosomes to IFs. For example, claudins
bind to the actin filaments via zona occludin-1,-2 and -3 (ZO-1, -2 and -3), E-cadherin is bridge to the
actin filament via catenins, and desmosomes to intermediate filaments via desmoplakin. It is important to
realize that intercellular adhesion do not just promote cell-cell adhesion, but also affects numerous cellular
processes such as morphogenesis, differentiation, migration, survival. The formation and dissociation of
intercellular junction is dynamically regulated by signal transduction pathways and extracellular matrix
elements to ensure proper development, growth and remodeling throughout life. In this course, we will
examine some of the key regulators of intercellular adhesion, such as nectins and nectin-like molecules,
intracellular complexes (Par3, Par6, aPKC complex, crumb complex and scribble complex), the Rho
family of small GTPases, and matrix remodeling metalloproteinases. It is also important to aware that
intercellular junctions, in particular TJs and AJs, concentrate several regulatory factors, transmembrane
receptors and oncogenes, which in turn have potent effects on cell dynamics.
Cross talk between intercellular adhesion systems
Studies indicate that the formation and maintenance of TJs and Desmosomes is dependent
on AJs. This raises the question as to how the assembly of intercellular junctions is orchestrated and
dynamically regulated. In the early 1990s, our understanding of the regulatory events controlling AJs
assembly was advanced by the discovery of the nectins and later afadin. The nectin-afadin system plays an
important role in promoting the formation of several intercellular junctions of a diverse group of cells.
The focus of this course will be on the promotion by the nectin-afadin system in promoting epithelial-
epithelial and interneuronal synapse adhesion.
Nectins are Ca -independent immunoglobulin (Ig)-like cell adhesion molecules (CAMs)
that stabilize the epithelium and endothelium and play key roles in establishing apical-basal polarity. The
nectin family is composed of at least four members that form homotypic and heterotypic associations
within the family, the nectin-like family and other transmembrane molecules. The focus of the lecture is
on the homotypic association of nectin-1 molecules and how it promotes the formation of adherens and
tight junctions. Intracellularly, nectins bind to afadin. There are two splice variants of afadin: a long form
(I-afadin) and a short form (s-afadin). It is the long form, I-afadin, which binds indirectly to the actin
cytoskeleton. For example, nectin-1 is linked to the actin cytoskeleton in three ways: (1) the binding to a-
catenin, (2) the ponsin–vinculin unit, and (3) the ADIP–a-actinin unit. While the focus of the lecture in on
the role of nectin-1 in promoting junctional complex formation, it is important to note that afadin is a
scaffold protein to which several adaptors bind, not just a direct interaction with nectin and F-actin. For
example, afadin binds to ZO-1, IQGAP and other proteins. As shown on two slides, unknown proteins
may regulate this cross-communication.
1 Nectin-1 is ubiquitously expressed by a broad spectrum of cells that include epithelial, fibroblast
and neural cells. The current model of nectin and cadherin dimerization is that they first form cis-
homodimers and then trans-homodimers (although some heterodimers between two nectin and two
cadherins do exit). The interactions were confirmed by a variety of molecular and imaging techniques.
Several studies indicate that nectin-afadin and E-cadherin systems are physically and
functionally associated. Much remains to be understood about the intracellular molecules that promote
their associations, as indicated by an X in the diagram shown in class.
Nectin-like molecules (Necls)
Like nectins, their N-terminal ectodomains form homotypic and heterotypic calcium-independent
associations with other nectins and necls. However, unlike nectins, necls do not bind at their C-terminals
to afadin. Instead, the PDZ binding motifsa bind to other adaptor molecules. Necls are not found at TJs
or AJs, but localize to the basolateral plasma membrane where they recruit membrane receptors (e.g α β3 V
integrin and platelet-derived growth factor receptor-PDGFR) to regulate cell motility and proliferation. I
will elaborate on the role of necls when we discuss epithelial to mesenchyme transformation and cell
motility in future lectures.
Regulation of junctional complex assembly
Takai and Nakanishi (2003) Nectin and afadin: novel organizers of the intercellular junctions. J. Cell
Science 116: 17-27. Most of the information below is verbatim from this review article.
Several lines of evidence indicate that the nectin-afadin and E-cadherin–catenin systems
are physically and functionally associated in epithelial cells. Firstly, during the formation of the junctional
complex involving AJs and TJs in Madin Darby canine kidney (MDCK) cells that stably express
exogenous nectin-1 (nectin-1-MDCK cells), E-cadherin is recruited to the nectin-1-based junctions where
afadin co-localizes. At the initial stage of formation of AJs and TJs, primordial spot-like junctions first
form at the tips of the cellular protrusions that radiate from adjacent cells. Components of the E-
cadherin–catenin and nectin-afadin systems, ZO-1 and JAM colocalize to these spot-like junctions, at
which neither claudin or occludin is concentrated. The spot-like junctions begin to fuse to form short line-
like junctions, at which claudin and occludin do accumulate. Although the precise timing of arrival of
each component at the junctional complex during its formation is unclear, a speculative model for
formation of the junctional complex is proposed.
In the model, all CAMs, including nectin, E-cadherin, JAM, claudin and occludin, are diffusely
distributed on the free surface of the plasma membranes of migrating cells. When the two migrating cells
contact through protrusions such as filopodia and lamellipodia, nectin and E-cadherin separately form
trans-dimers that form micro-clusters at intercellular contact sites. Because, kinetically, nectin forms
micro-clusters more rapidly than E-cadherin, the nectin-based micro-clusters are mainly formed at the
initial stage. The nectin-based microclusters then recruit E-cadherin, which results in the formation of a
mixture of nectin- and E-cadherin based microclusters. The nectin and E-cadherin molecules in these
microclusters are associated through afadin and catenins that are linked to the actin cytoskeleton. E-
Cadherin-based micro-clusters that form slowly and independently of the nectin-based microclusters
rapidly recruit the nectin-afadin complex to form other primordial spot-like junctions. These primordial
junctions fuse with each other to form short line-like junctions, which develop into more matured AJs.
During the formation of AJs, JAM is first assembled at the apical side of AJs, and this is followed by the
recruitment of claudin and occludin presumably through ZO-1, ZO-2 and ZO–3, which eventually leads to
the establishment of claudin-based TJs. There is evidence that the formation of TJs and AJs also sets the
stage for the formation of desmosomes.
2 Small monomeric GTPases: the Rho-family
The Rho-family of small monomeric GTPases: a complex and diverse family of molecular
switches. The family is part of a larger Ras superfamily of monomeric GTPases. Alberts et al. Pages
The Rho family of small monomeric GTPases is defined by the presence of structural
similarity to a Rho-type GTPase-like domain. A subset of the Rho GTPase, RhoA/Rho1, Rac1 and
Cdc42, play critical and distinctive roles in regulating actin-cytoskeletal dynamics and from which most of
our functional information about small GTPases are derived. Although, the focus is the involvement of
Rho GTPases in regulating microfilament dynamics, it is important to be aware that they also promote cell
growth, vesicular traffic, have anti-apoptotic functions as well as regulating gene expression.
Rho GTPases are relatively small enzymes with one GTPase domain, and short N and C-
terminals. The GTPase domain binds GDP and GTP with high affinity, cycling between active GTP-
bound and inactive GDP-bound states. Hydrolysis of GTP to GDP + Pi induces a conformational change
that inactivates Rho factors.
RhoA signaling and stress fiber formation
RhoA was first implicated in the formation of stress fibers and focal adhesion, two critical
events for the forward mo