Since their discovery in the early 1990's, the primary focus of aquaporin research has been on the transcellular transport of water and other small solutes across the cell membrane. Novel roles for aquaporins besides bulk transport of water have been identified. Saadoun et al 
first proposed roles for AQP1 in cell migration and angiogenesis. Since then, other aquaporins have been identified to have similar roles in cell migration and proliferation 
. However, even these novel roles are thought to occur via local water flux driven by small osmotic changes due to actin polymerization-depolymerization and transmembrane ion fluxes 
. Others have proposed that AQP0 could play a role in modulating cell-cell contacts, potentially via interaction with connexins 
. While AQP4 has also been postulated to participate in mediating cell-cell contacts 
, this has been met with some controversy 
. To our knowledge, in this report we provide the first description of a role for an aquaporin in directly associating with and modulating the cytoskeleton.
Twelve mammalian homologues have been identified, with distinct cellular and subcellular distributions 
. It has been hypothesized that different homologues are required to achieve distinct regulation of water homeostasis in different cells and organs 
. In addition, localization and regulation of each aquaporin is distinct, and therefore allows for further fine-tuning of water regulation in cells. However, our study demonstrates an additional rationale for multiple aquaporins. In addition to its role in water transport, AQP5 directly binds to MTs and increases their assembly. This function is at least relatively specific, since AQP1 did not alter MT dynamics. Our data indicates that AQP5 increases MT assembly primarily by stabilizing MTs, but in addition, our cell-free assay indicates that AQP5 also can promote MT nucleation. It is not known if this latter mechanism occurs in vivo
Our data shows that the carboxyl-terminus– the longest intracellular portion of the protein– is sufficient to mediate this increase in MT assembly. The carboxyl-terminus is distinct from the conserved segment of the aquaporins, the Asn-Pro-Ala (NPA) motif, that are critical in regulating water transport through the pore and through the membrane 
. When the carboxyl-terminus of AQP5 and AQP1 are aligned, only 11/35 (32%) amino acids are identical, so it is perhaps not surprising that that the two proteins do not function similarly when it comes to MT polymerization.
The fact that the carboxyl-terminus is sufficient to allow for MT assembly raises other interesting considerations. While aquaporins are present in the membrane as tetramers, the water channel forms through the center of the monomer, rather than the center of the tetramer, as is often observed in ion channels 
. Structurally, based on analysis of AQP1, each monomer is positioned such that the outside face of the tetramer is hydrophobic, and the center of the tetramer is hydrophilic 
. Further, in AQP5, a lipid occludes the putative central pore 
. Given the tetrameric structure, one might speculate whether this organization allows for juxtaposition of four AQP5-CT domains, further enhancing AQP5 effects on MT stabilization.
Previous work from our lab and others has shown that AQP5 modulates paracellular permeability in epithelial cells 
. This is associated with changes in proteins mediating cell-cell contacts. However, the mechanisms by which changes in an apically expressed water channel alter proteins in the lateral membrane are not clear. Our study provides insight into one potential mechanism, by showing that AQP5 can directly modulate MT stability. Further molecular definition of how altered MT assembly affects adherens junction and desmosomal proteins awaits elucidation and these responses in cell-cell contacts maybe cell-type specific. Other microtubule associated proteins such as MAP4 
are present in airway epithelial cells, suggesting that MAP4, along with AQP5, could modulate airway epithelial microtubule dynamics. However, AQP5 is tightly regulated in lung epithelial cells, and dynamically responds to several physiologic and pathologic stimuli including TNFα 
, cAMP 
, osmotic stress 
, LPS 
and shear stress 
. While it has been hypothesized that tight regulation of AQP5 may be needed to control transmembrane water flux, however, coordination of MT dynamics with consequential changes in paracellular permeability is an alternate explanation for this level of regulation. AQP5 can be internalized in response to certain stimuli such as cAMP in as little as two minutes 
and be degraded in response to osmotic stress in thirty minutes 
. Clearly, AQP5 is subjected to multiple levels of regulation, leading to changes in paracellular permeability on different time-scales in response to different types of luminal stimuli. While our study indicates that AQP5 can directly mediate changes in microtubule dynamics, we do not rule out the possibility of subsequent indirect effects on microtubule polymerization also leading to the changes in MT stability. To our knowledge, this is the first demonstration that an aquaporin can directly mediate changes in cytoskeletal organization via a mechanism independent of water transport, providing yet another novel role for an aquaporin.