PH is a congenital brain disorder thought to involve disruption of initial neuronal migration (Lu and Sheen, 2005
). The current work suggests that two genes FLNA
causal for this disorder interact to disrupt neural migration and the integrity of the neuroependyma. Big2 regulates the phosphorylation state of FlnA and over-expression of phosphoFlnA impairs neural migration into the cortex. FlnA phosphoryation state dictates actin binding affinity and alters the focal adhesion sites, thereby providing a potential mechanism for regulation of neural migration and PH formation.
The overlapping defects in humans and mice harboring FLNA
gene mutations indicate some shared molecular function across various organ systems. Humans harboring ARFGEF2
mutations show microcephaly and PH (Sheen et al., 2004
), and the Arfgef2−/−
mouse has variable findings of PH as well as exencephaly and omphalocele formation. A prior study reported early embryonic lethality of a gene trap mouse with disruption of the Arfgef2
gene (Grzmil et al., 2011
). In an analogous fashion, human FLNA
mutations lead to milder microcephaly and PH, as well as midline cleft palates, sternal clefts, omphaloceles, and encephaloceles (Robertson et al., 2003
; Robertson, 2005
; Gerard-Blanluet et al., 2006
; Sole et al., 2009
). The null FlnA
mice are embryonic lethal and therefore do not develop PH, but they do show a disrupted neuroependymal lining, microcephaly, and midline defects including omphalocele formation (Lian and Sheen, in press). Taken together, these findings begin to implicate FLNA
genes in a shared molecular function regulating progenitor development.
BIG2 appears to regulate various modalities involved in neuronal migration. Our prior studies have shown that inhibition of BIG2 through BFA leads to disruption of the neuroependymal lining and heterotopia formation (Ferland et al., 2009
). In the current work, Arfgef2−/−
mice show the same disruption of the nestin-positive radial glial scaffolding and of the positioning of various neuronal progenitors directly within the ventricular zone. In this context, disruption of glial-guided migration from loss of neuroependymal integrity and consequent disruption of radial glia might be sufficient to cause PH. Exposure of E14.5 Arfgef2−/−
mice to BrdU and analyses at E18.5 revealed more cells within the IZ, suggesting a disruption in the intermediate stages of migration. These observations, however, would suggest that Big2 plays an additional role in intrinsic cell migration or motility. Thus, PH likely arises from a contribution of both cell intrinsic (neural migration) and cell extrinsic (disruption of the neuroependyma).
The mechanism by which loss of Big2 function leads to increased FlnA and phosphoFlnA expression is unclear. It is unlikely that the A-kinase anchor protein sites from Big2 directly assist in FlnA phosphorylation, given that loss of Big2 function leads to upregulation and not downregulation of FlnA phosphorylation. Both Big2 and FlnA, however, have been implicated in vesicle trafficking of receptors along the cell membrane, suggesting that they regulate the stability and clearance of various proteins along the cell surface (Liu et al., 1997
; Lin et al., 2001
; Charych et al., 2004
; Shen et al., 2006
; Ravid et al., 2008
). To address whether loss of Big2 impaired protein degradation or enhanced protein synthesis, we quantified the clearance of FlnA and β-integrin protein levels in Arfgef2−/−
progenitor cells at different time points after exposure to cycloheximide, an inhibitor of protein synthesis. Both proteins showed a progressive decline in expression by western blotting after two hours in WT precursors. However, we observed a trend toward increased rates of clearance for FlnA in the WT as opposed to Arfgef2−/−
cells, albeit this was not statistically significant (data not shown). The rate of degradation of β-integrin was significantly slower in the null Arfgef2
cells, suggesting that Big2 did regulate the stability of certain receptors near the cell surface. The greater stability of the actin binding FlnA might require a longer time-frame for clearance than could be performed in the current studies to observe a significant change (due to cyclohexamide toxicity effects). Alternatively, phosphorylation of FlnA at ser2152 has been shown to direct various proteins toward the cell membrane (Vadlamudi et al., 2002
), and we had also previously observed that dominant negative inhibition of Big2 impaired the delivery of FlnA to the cell membrane (Lu et al., 2006
). Thus, the upregulation of phosphoFlnA might serve as a compensatory response by cells to promote FlnA function at the cell membrane. Further studies will be necessary to address these possibilities.
The neuropathological abnormalities observed in our current work are remarkably similar to those seen in the null Mekk4 mouse (Sarkisian et al., 2006
), suggesting a shared common endpoint. Mekk4 suppression leads to heterotopia formation in mice, disruption of the neuroependyma, and is associated with an increase in both FlnA and phosphoFlnA levels. MEKK4 is a MAP3Ks that affects the activity of downstream MAP2Ks and MAPKs including JNK and p38 (Gerwins et al., 1997
). Integrin mediates the phosphorylation of JNK and directs cell spreading and adhesion, and this can be abolished by BFA (Nguyen et al., 2000
). We also observed an increase in integrin expression with loss of Big2 (data not shown). Taken collectively, FlnA interactions with both Big2 and surface receptors such as integrins suggest that it serves to integrate the signaling and activation of the JNK pathway, in part through Mekk4.
FlnA phosphorylation state may serve as a key regulator in neural migration. Several studies have demonstrated that both increased and decreased levels of FlnA phosphorylation (ser2152) impair migration. Growth factor mediated phosphorylation of FlnA (ser2152) leads to increased migration of melanoma cells (Woo et al., 2004
; Ravid et al., 2008
). Conversely, mice deficient in Mekk4 showed both increase in FlnA and phosphoFlnA expression, and impaired neuronal migration (Sarkisian et al., 2006
). Based on these observations and prior reports that phosphoFlnA impaired calpain dependent degradation of FlnA (Sarkisian et al., 2006
), Sarkisian et al. raised the possibility that phosphoFlnA prevented FlnA degradation and led to increased FlnA levels, thereby inhibiting migration. Our current work, however, suggests that the phosphorylation state of FlnA may play a more pivotal role in impairing migration. In utero
electroporation of phosphomimetic FlnA-S2152D impairs migration, whereas the phosphodeficient FLNA-S2152A enhances migration. Moreover, consistent with prior reports that FlnA phosphorylation reduces the binding of FlnA to actin filaments in general (Ohta and Hartwig, 1995
), we find that phosphorylated FlnA (ser2152) does not bind actin as strongly and leads to redistribution of FlnA to the cytoplasm. Overall, a balance in FlnA phosphorylation at ser2152 may be pivotal in controlling migration. For example, increased FlnA phosphorylation is thought to impair actin binding and consequent formation and/or turnover of actin networks, which would presumably disrupt migration (Ohta and Hartwig, 1995
). On the other hand, phosphorylation also prevents FlnA degradation by calpain cleavage and increased FlnA levels should promote migration by enhancing actin network formation/turnover (Garcia et al., 2006
; O’Connell et al., 2009
Focal adhesions serve as the mechanical linkages between the extracellular matrix and intracellular actin cytoskeleton, thereby directing signaling proteins at sites of integrin (and filamin) binding and clustering. Their assembly at the leading edge and disassemby at the rear of cells are essential for migration (Vicente-Manzanares et al., 2009
). Prior studies have suggested that increased cell motility is associated with smaller but more numerous focal adhesions (Xu et al., 1998
; Ziegler et al., 2006
). We similarly observed an increase in neural migration following over-expression of the FLNA-S2152A construct, which is associated with smaller but more numerous paxillin associated focal adhesions. Conversely, larger and fewer paxillin associated focal adhesions were seen in phosphomimetic FLNA-S2152D transfected cells and led to a reduction in migration. It remains unclear whether and how the phosphorylation dependent FlnA-actin binding affinity regulates focal adhesion sites. One possibility is that the increased binding affinity (FlnA unphosphorylated state) is more permissive to signal transduction and allows for FlnA and actin dependent, dynamic changes, which may facilitate paxillin containing focal adhesion remodeling. Similarly, the phosphomimetic mutant weakens FlnA and actin interaction which may in turn limit actin cytoskeletal conformational or structural changes and impair focal adhesion disassembly required for migration. Nevertheless, both alterations in actin binding and increased size of paxillin focal adhesion sites could be detrimental to neural migration.
The current study uncovers an important functional link between the actin-binding FlnA and vesicle trafficking related Big2 proteins in guiding neural migration, presumably through Big2-dependent regulation of FlnA phosphorylation and association with actin-focal adhesion organization. It is important to recognize that FlnA-dependent regulation of the actin cytoskeleton will not only influence the cell shape and movement, but may also govern other actin-dependent processes, namely vesicle trafficking. It will be of interest to address whether FlnA regulates expression and function of vesicle-associated proteins such as Big2, and whether disruption of this pathway also alters paxillin or other focal adhesion complexes. In this context, interactions between proteins that regulate vesicle formation (i.e. gefs) and actin stability/turnover (i.e. filamins) may begin to provide a means by which changes in intracellular vesicular trafficking can give rise to the various neurological phenotypes associated with PH, including disruption of the neuroependymal lining, reduction in brain size, and impairment in neuronal migration.