Filamin A and Big2: A shared endocytic pathway

Abstract

Neural proliferation, migration and differentiation require reorganization of the actin cytoskeleton and regulation of vesicle trafficking to provide stability in maintaining cell adhesions, allow for changes in cell shape, and establishing cell polarity. Human disorders involving the actin-binding Filamin A (FLNA) and vesicle trafficking Brefeldin-associated guanine exchange factor 2 (BIG2 is encoded by the ARFGEF2 gene) proteins are implicated in these various developmental processes, resulting in a malformation of cortical development called periventricular heterotopia (nodules along the ventricular lining) and microcephaly (small brain). Here we discuss several recent reports from our laboratory that demonstrate a shared role for both proteins in actin-associated vesicle trafficking, which is required to maintain the expression and stability of cell adhesion and cell cycle associated molecules during cortical development. While changes in FLNA and BIG2 have first been linked to disorders involving the central nervous system, increasing reports suggest they are associated with aberrant development of various other organ systems in the body. These studies suggest that vesicle trafficking defects in FLN-GEF dependent pathways may contribute to a much broader phenotype than previously realized.

Introduction

Dynamic changes in the actin cytoskeleton are required for fundamental processes that drive cell adhesion and migration.^1,2 Filamins are actin-binding proteins that regulate the cytoskeleton by serving as a scaffold for stabilizing actin filaments and linking them to the cell membrane to allow for signal transduction.³ Filamin A (FLNA) is one of three isotypes (the others being FLNB and FLNC) and has been most intensely studied. FLNA is a 280 kD protein that undergoes both homo and heterodimerization with other filamins.⁴ It is comprised of an actin-binding N terminus, followed by 24 Ig-like repeats with β-pleated sheet units. The end C-terminal repeat 24 is responsible for dimerization, whereas repeats 16–23 largely mediate binding to over 90 interacting partners.¹ In general, FLNA effects have largely been thought to occur through its regulation of adhesion molecules/receptors and changes in actin filaments, predominantly through actin effectors such as the RhoGTPases. The cell biological changes observed following disruption of filamin with the various interactors have not necessarily translated into human disease phenotypes nor have they given insight into which pathways are most physiologically relevant. Here we focus on a FLNA-associated human and mouse disease phenotypes in providing insight into actin-associated processes that would contribute to this disorder and review recently discovered interactions between FLNA and another protein BIG2, causal for the same disease, in effecting membrane trafficking and subsequent cell adhesion, migration, and proliferation.

FLNA and BIG2 in Human Malformations of Cortical Development

Characterization of the human phenotype in disorders associated with FLNA mutations provides a means with which to identify the most relevant FLNA-dependent biological pathways. Loss of FLNA function leads to periventricular heterotopia (PH), a malformation of cortical development characterized by ectopic nodules of neurons along the neuroependyma of the lateral ventricules. During normal development, proliferating neural progenitors along the neuroependyma undergo a decision pathway to become post-mitotic neurons/intermediate progenitors or to remain as a primary progenitor. With transformation to the intermediate or post-mitotic state, neural cells migrate from the ventricular zone to the cortical plate. Failure in initial migration due to FLNA-dependent defects in the actin cytoskeleton was initially thought to cause PH.⁵ FLNA dependent PH is an X-linked disorder such that heterozygous females harbor the PH phenotype, and males are generally embryonic lethal. Loss of FLNA function leads to cell autonomous defects in neural motility, although many cells reached their appropriate destination in the cortical plate.^6,7 These findings raised the possibility that some fraction of neural progenitors harboring the mutant FLNA did not migrate properly whereas those expressing the normal FLNA protein had no migratory impairment.

In addition to a motility problem, recent studies suggest that FLNA loss leads to a proliferative defect. While microcephaly is not seen in heterozygous females with FLNA mutations (likely due to mosaicism), hemizygous males have been reported to have thinner cortices, and loss of cortical convolutions consistent with underlying microcephaly.⁸ Loss of FlnA function in homozygous mice leads to microcephaly and a reduction in neural progenitor proliferation over time.⁹ Characterization of null FlnA mouse progenitors has shown that the decreased proliferation is not due to an increase in cell death, but rather a decrease in production due to a prolongation in the cell cycle time of progression through G2-M and G1-S phases. FlnA inhibition leads to impaired degradation of several cyclinB/cdk1 associated proteins. A slowing in the cell cycle ultimately causes a delay and reduction in progenitor production over time. Moreover, M phase (mitosis) occurs along the neuroependymal lining, suggesting that a common process might mediate neuroependymal integrity, initial migration and brain size.

While coordinating the actin cytoskeleton through FLNA assists in many basic cell processes, identification of a second gene causal for PH suggest that disruption of actin filaments alone is not responsible for the disease phenotype. Autosomal recessive mutations in the human gene ADP-ribosylation Factor Guanine Exchange Factor 2 (ARFGEF2, encodes for BIG2) also cause PH and microcephaly.^10-12 Brefeldin A guanine exchange factor 2 (BIG2) is a 180 kDa protein which belongs to a family of three highly conserved large molecular weight mammalian Sec7-GEFs (the others being GBF1, BIG1/2), characterized by their sensitivity to the fungal metabolite Brefeldin-A (BFA).^13-15 A highly conserved Sec7 domain in these GEFs directs GDP-to-GTP exchange and activation of the ADP-ribosylation factors (ARFs).^16-20 ARFs regulate vesicular traffic through recruiting coat proteins.²¹ The N-terminal of BIG2 binds Exo70, a member of the exocyst complex involved in vesicle exocytosis.²² Several AKAP (A-kinase anchoring protein) binding sites are located within the Exo70 binding region. BIG2 residues 27–48 interact with PKA (Protein Kinase A) subunits RIα and RIβ residues 284–301 interact with subunits RIlα and RlIβ, and residues 517–538 interact with subunits RIα, RIlα, and RlIβ.²³ Finally, at its C-terminal, BIG2 has been shown to bind the β subunit of GABA receptors.²⁴ As a kinase anchoring protein, BIG2 is implicated in the spatiotemporal activation of cAMP signaling, and PKA-dependent phosphorylation also appears to regulate BIG2 activation of the ARFs.^25,26 Given that vesicle trafficking is in part mediated through the actin cytoskeleton, some interplay between these two genes is likely most relevant in disease pathogenesis and understanding their primary functions.

Comparisons between the human disease phenotypes and animal models associated with the two causal PH genes provide several key insights. First, PH due to ARFGEF2 mutations is an autosomal recessive disorder, indicating that all the neural progenitors harbor the genetic mutation.¹¹ Thus a cell-autonomous migratory defect alone from disruption in actin-dependent cell morphology is not sufficient to explain PH formation since many neurons in this disorder migrate appropriately into a normal appearing, overlying cortex. Recent studies suggest that loss in cell-cell adhesion along the neuroependyma leads to a disruption in ventricular neuroepithelium, alters radial glial fibers, and disrupts migration out of the ventricular zone.^27-29 This impairment in cell-cell adhesion is likely a primary cause of the heterotopia. Both loss of FlnA and Big2 function in mice, however, also demonstrate impairments in neural motility.^30,31 A migratory defect might be more pronounced near the end of cortical development as a slower migratory rate might influence the ability of the latest born neurons to reach their final destination. In this context, shared biological mechanisms that regulate cell adhesion and cell migration in progenitors along the neuroepithelial lining are likely most relevant for discerning FLNA and BIG2 function. Second, both genes also lead to microcephaly in mice.^9,31 The impairment in degradation of various cell-cycle associated proteins seen with loss of FlnA function would be indicative of a problem in vesicle trafficking, involving endocytosis and/or lysosomal degradation. Third, both proteins show strong overlapping expression along the neuroependymal lining.³² Thus, a trafficking defect within progenitors along the neuroepithelium could disrupt the lining (from loss in cell-cell contacts), disrupt initial migration (from loss in cell-ECM contacts) and alter proliferation (from impaired trafficking of cell receptors and intracellular signaling molecules).

Endosomal Trafficking in Phenotypes Associated with PH

Several studies have implicated BIG2 and FLNA in endosomal vesicle trafficking in cortical development. Although BIG2 is mainly associated with trafficking through the trans-Golgi network, it also regulates recycling endosomes through class1 ARFs.³³ ARFs have been shown to reside at the cell surface with ARF1 and ARF3 mediating endocytosis.^21,34 Further, BIG2 dependent recycling of the integrin receptor at the cell membrane has been thought to mediate neuronal migration.³⁵ While FLNA has been linked to multiple cell functions (receptors, intracellular signaling pathways), the actin binding protein is directly involved in trafficking. FLNA binds to the protease furin, can direct the rate of internalization of this protein, and directs sorting of furin from the early endosome to the trans-Golgi network. FLNA also is required for appropriate localization of late endosomes and lysosomes.³⁶ Mechanistically, furin co-localizes with caveolin, an integral membrane protein involved in receptor independent endocytosis.³⁷ Caveolin binds FLNA, and FLNA phosphorylation and its binding to actin are required for caveolin dependent endocytosis.^38-40 Phosphorylation of caveolin is involved in integrin-regulated caveolae trafficking and also in signaling at focal adhesions in migrating cells.⁴¹ Transient inhibition of integrin binding and signaling disrupts the attachment of apical processes along the ventricular lining, alters internuclear kinetic migration and alters the cleavage orientation of dividing cells.⁴² Collectively, these studies provide a common endocytic pathway between FLNA and BIG2 in causing the PH phenotypes.

Endocytosis relies on vesicle formation for maintenance of protein localization, expression, and function, and actin for vesicle movement.^43,44 A fundamental question lies in whether FLNA and BIG2 act separately or in some coordinated fashion during endocytosis. Our recent studies have shown that Big2 and FlnA physically interact and both are expressed in the Golgi and along the cell membrane.^31,32 Loss of Big2 in transgenic mice results in a consequent increase in both FlnA and phosphoFlnA at serine 2152. FlnA overexpression impairs neural migration by altering the number and size of paxillin focal adhesions. Conversely, loss of FlnA expression similarly impairs neural migration, in part, through changes in paxillin stability at the cell membrane. Further, both enhanced or loss of FlnA expression has been shown to cause PH in humans.^5,45 Taken in this context, Big2 could alter FlnA stability and expression through endocytosis, raising the possibility that FlnA alone serves as a final common pathway in this disease.

More recent studies provide another potential explanation for FlnA-Big2 interactions. Loss of FlnA leads to a reciprocal, enhanced expression of Big2, suggestive of a compensatory feedback.³⁰ We have also found that Big2 localization is dependent on FlnA, with FlnA phosphorylation leading to redistribution of Big2 from the Golgi apparatus to the membrane. Arf1 activation through Big2 at the membrane is dependent on FlnA. Thus, loss of FlnA not only affects migration, cell adhesion and proliferation by disrupting formation of actin filaments, but also by indirectly disrupting vesicle trafficking through Big2-dependent Arf1 activation. The increase in Big2 following FlnA loss would be consistent with cellular attempts to promote Big2 redistribution to the membrane to activate Arf1. Further, the increase in FlnA and phospho-FlnA after Big2 loss would reflect a cellular response, favoring Big2 localization to the cell periphery and Arf1 activation. Overall, these studies suggest that disruption in vesicle trafficking and endocytosis with indirect consequential effects on cell-cell and cell-ECM molecules are primarily responsible for the PH phenotypes.

Open Questions

While Big2 activation leads to Arf1 dependent formation of vesicles, it is unclear whether and how actin cytoskeletal changes through FlnA actually regulate vesicle trafficking. FlnA does bind the Rho GTPases which direct actin cytoskeletal changes and have been shown to mediate endocytosis.^46,47 The Rho GTPases comprise a small family of G proteins that regulate actin dynamics, Cdc42, Rac1, and RhoA being the most studied. They interact with over 60 effectors, some of which are involved in actin nucleation and vesicle trafficking. None, however, have been specifically linked to FlnA. Identification of a shared Rho-FlnA effector is necessary to further elucidate the disease-causative pathway.

Although PH involves altered cell-cell adhesion effecting the neuroependymal lining and cell migration, other studies suggest that disruption of either Big2 or FlnA impairs proliferation. Loss of FlnA causes changes in the rate of degradation of several cell cycle proteins involved in G2-M and to a lesser degree G1-S phases of the cell cycle. These findings suggest that PH genes are likely involved in progression from endocytosis to lysosomal degradation. This possibility is consistent with the finding that the localization of both late endosomal and lysosomal compartments is dependent on FlnA.³⁶ Finally, mutations in two additional genes, atypical cadherin receptor ligands (involved in cell cell adhesion) FAT4 and DCHS1 have recently been shown to cause periventricular heterotopia, with nodules along the lateral ventricle and altered progenitor proliferation.⁴⁸ FAT and DCHS have been implicated in the Hippo signaling pathway, which controls organ size through regulation of cell proliferation and apoptosis through cyclin E (Cdk2) during G1-S phase.^49,50 It will be of interest to see whether the regulation of these receptors follows a FLNA and BIG2 dependent pathway from endocytosis to lysosomal degradation.

Conclusion

Our studies provide a hypothetical model for PH formation (Fig. 1). First the primary anatomical defect leading to the PH phenotype (loss in neuroependymal integrity, impaired migration, and reduced proliferation) occurs within neural progenitors along the neuroepithelial lining. Genes implicated in PH (FlnA, Big2) regulate the stability, turnover and degradation of cell adhesion molecules (β-catenin, N-cadherin, Fat4, Dchs1) and cell-ECM receptors (integrins, paxillin adaptor proteins). FlnA phosphorylation targets Big2 to the membrane, allowing for Arf-dependent activation and vesicle formation. Endocytosis occurs through a caveolin dependent mechanism leading to internalization of catenin/cadherin/protocadherins and integrins. Specificity for some of these molecules extends from their binding of filamins. The downstream mechanisms that mediate endosomal processing in contributing to PH are not known, and remain a focus of continued research. Lastly, human phenotypes associated with mutations in FLNA and ARFGEF2 extend beyond neurological disorders. Disruption of FLNA leads impairments in virtually every organ system, from skin to skeleton, whereas BIG2 defects have been associated with obstructive cardiomyopathy, recurrent infections and feeding difficulties.^10,51 The increasingly broad genotype-phenotype correlations reiterate the significant role actin and vesicle trafficking play in human development and disease.

Figure 1.

Endosomal recycling through Big2 and FlnA. FlnA phosphorylation localizes Big2 to the cell membrane, thereby activating the Arfs. Arfs are required for vesicle formation. Disruption of endocytosis leads to disruption of cell-cell adhesion molecules, giving rise to periventricular heterotopia. Impaired turnover of integrin receptors alters neural migration. Impaired signaling through cadherin/catenins alters neural proliferation.

Zhang J, Neal J, Lian G, Hu J, Lu J, Sheen V. Filamin A regulates neuronal migration through brefeldin A-inhibited guanine exchange factor 2-dependent Arf1 activation. J Neurosci. 2013;33:15735–46. doi: 10.1523/JNEUROSCI.1939-13.2013.

Zhang J, Neal J, Lian G, Shi B, Ferland RJ, Sheen V. Brefeldin A-inhibited guanine exchange factor 2 regulates filamin A phosphorylation and neuronal migration. J Neurosci. 2012;32:12619–29. doi: 10.1523/JNEUROSCI.1063-12.2012.

Lian G, Lu J, Hu J, Zhang J, Cross SH, Ferland RJ, Sheen VL. Filamin A regulates neural progenitor proliferation and cortical size through Wee1-dependent Cdk1 phosphorylation. J Neurosci. 2012;32:7672–84. doi: 10.1523/JNEUROSCI.0894-12.2012.

10.4161/bioa.28516