4.3 Afadin
Afadin is a filamentous actin binding protein with two Ras domains,
a forkhead association domain, a dilute domain, a PDZ domain,
and three proline-rich domains. Afadin is implicated in many cellular processes from cell survival, cell proliferation to cell migration
and formation of the apical junctions in epithelial cells [80–83]. Afadin can interact both directly or indirectly with the actin cytoskeleton through several partners such as JAM-A, ZO1/ZO2, vinculin,
and α-actinin [84–88]. Afadin is thus a strong candidate to be
involved in mechanoregulated processes as many of its partners
have been already linked to mechanotransduction. Interestingly,
depletion of Afadin, JAM-A or double depletion of ZO1/ZO2
results in similar phenotypes with increased contractility triggered
by activation of RhoA and phosphorylation of myosin-light chain
[89–91]. In response to ZO1/ZO2 double depletion, Afadin is
recruited to the cellular cell contacts, and inhibiting contractility
perturbs the homogeneous localization of Afadin. Thus, myosin
contractility is essential for maintaining Afadin uniform distribution
along the zonula adherens (ZA). Removing Afadin in ZO1/ZO2
depleted cells specifically altered actomyosin architecture at the ZA
of tricellular junctions where actin cables are anchored. This perturbation is accompanied by discontinuities in the E-cadherin,
claudin, and occludin stainings [92]. Taken together, this data
shows a synergy between ZO and Afadin depletions in disrupting
tissue integrity under tension. Afadin may therefore act as a robust
protein scaffold that maintains ZA architecture at tricellular
junctions.
JAM-A, a component of the TJs, via its PDZ binding motif, can
associate with signaling molecules such as scaffold PDZ proteins,
ZO1/2, and Afadin, as well as with the guanine exchange factor
PDZ-GEF2 [83, 93]. Monteiro et al. demonstrate that JAM-A
interacts with Afadin, ZO2, and PDZ-GEF1 to activate the small
GTPase Rap2c [89]. The activation of Rap2c controls the contraction of the apical cytoskeleton regulating the epithelial permeability
to prevent cell injury. In this study, the authors also mention that
Afadin is able to immunoprecipitate a doublet of JAM-A that might
represent phosphorylated forms of JAM-A, although they did not
investigate this phosphorylation but another team did. Scott at al
show that mechanical stimulation can trigger phosphorylation of
JAM-A [94]. Forces were applied by using fluid shear stress or by
using magnetic tweezer. After applying forces, several biochemical
analyses were performed that demonstrate that tension induces
rapid phosphorylation of JAM-A S284. This phosphorylation of
JAM-A induces activation of RhoA by PKCζ triggering cell stiffening by modifying actin cytoskeleton. These results clearly demonstrate that JAM-A is a direct transducer of mechanical forces.
Interestingly, when JAM-A is localized at the TJs which are under
high levels of tension [95] it is phosphorylated [96]. It is thus
tempting to speculate that mechanical tension, above a certain
266
Elsa Bazellie ` res and Andre ´ Le Bivic
Afadin is a filamentous actin binding protein with two Ras domains,
a forkhead association domain, a dilute domain, a PDZ domain,
and three proline-rich domains. Afadin is implicated in many cellular processes from cell survival, cell proliferation to cell migration
and formation of the apical junctions in epithelial cells [80–83]. Afadin can interact both directly or indirectly with the actin cytoskeleton through several partners such as JAM-A, ZO1/ZO2, vinculin,
and α-actinin [84–88]. Afadin is thus a strong candidate to be
involved in mechanoregulated processes as many of its partners
have been already linked to mechanotransduction. Interestingly,
depletion of Afadin, JAM-A or double depletion of ZO1/ZO2
results in similar phenotypes with increased contractility triggered
by activation of RhoA and phosphorylation of myosin-light chain
[89–91]. In response to ZO1/ZO2 double depletion, Afadin is
recruited to the cellular cell contacts, and inhibiting contractility
perturbs the homogeneous localization of Afadin. Thus, myosin
contractility is essential for maintaining Afadin uniform distribution
along the zonula adherens (ZA). Removing Afadin in ZO1/ZO2
depleted cells specifically altered actomyosin architecture at the ZA
of tricellular junctions where actin cables are anchored. This perturbation is accompanied by discontinuities in the E-cadherin,
claudin, and occludin stainings [92]. Taken together, this data
shows a synergy between ZO and Afadin depletions in disrupting
tissue integrity under tension. Afadin may therefore act as a robust
protein scaffold that maintains ZA architecture at tricellular
junctions.
JAM-A, a component of the TJs, via its PDZ binding motif, can
associate with signaling molecules such as scaffold PDZ proteins,
ZO1/2, and Afadin, as well as with the guanine exchange factor
PDZ-GEF2 [83, 93]. Monteiro et al. demonstrate that JAM-A
interacts with Afadin, ZO2, and PDZ-GEF1 to activate the small
GTPase Rap2c [89]. The activation of Rap2c controls the contraction of the apical cytoskeleton regulating the epithelial permeability
to prevent cell injury. In this study, the authors also mention that
Afadin is able to immunoprecipitate a doublet of JAM-A that might
represent phosphorylated forms of JAM-A, although they did not
investigate this phosphorylation but another team did. Scott at al
show that mechanical stimulation can trigger phosphorylation of
JAM-A [94]. Forces were applied by using fluid shear stress or by
using magnetic tweezer. After applying forces, several biochemical
analyses were performed that demonstrate that tension induces
rapid phosphorylation of JAM-A S284. This phosphorylation of
JAM-A induces activation of RhoA by PKCζ triggering cell stiffening by modifying actin cytoskeleton. These results clearly demonstrate that JAM-A is a direct transducer of mechanical forces.
Interestingly, when JAM-A is localized at the TJs which are under
high levels of tension [95] it is phosphorylated [96]. It is thus
tempting to speculate that mechanical tension, above a certain
266
Elsa Bazellie ` res and Andre ´ Le Bivic