RESEARCH
Overview
Understanding the way major cellular systems integrate to control cell physiology and tissue function is at the forefront of Cell Biology.
Of particular importance is the interface between cell polarity, structure, and growth control, which must be finely tuned during morphogenesis to create organs and organisms. Spectrins are giant cytoskeletal proteins with two contrasting activities: They are best known for their ability to form fishing net-like structural networks with F‑actin at the cell cortex. However, they have also been shown to modulate protein trafficking in the endomembrane system, where they influence recycling/degradation decisions. The balance between these activities is unknown, and cell-type specific.
βH-spectrin (βH) is an emerging integrator of apicobasal polarity and growth in epithelia, where βH binds to two conserved regulators – the apical determinant Crumbs, and the Hippo[MST] pathway activator Expanded – facilitating Crumbs inhibition of growth. Both networking and trafficking roles of βH are implicated in this activity, exemplifying this balance. βH also has roles in nuclear envelope structure which are just emerging.
Introduction
Our research seeks to answer fundamental questions about the roles of the cytoskeleton at the cell membrane in epithelial cells – a nexus of interactions between cell structure (cytoskeleton, cell adhesion, morphogenesis), protein trafficking (endo- and exocytosis), and regulation (cell signaling, apicobasal polarity, growth).
Drosophila is our chosen model system because of the multidisciplinary combination of tools available, and because of its well-characterized development. We use biochemical, molecular and cellular techniques as well as classical and transgenic genetic approaches as appropriate.
The spectrin-based membrane skeleton (SBMS) is a ubiquitous structure that is found in all metazoan organisms. Spectrins are long heterotetramers of two α and two β chains, which crosslink F-actin to form two dimensional spectrin networks form in association with various cell membranes where they can modulate cell shape, integrity, and growth, as well as protein trafficking and nuclear morphology. Typically, different spectrin isoforms are polarized to distinct membrane domains. Drosophila provides a simple model system for examining this molecular scaffold, since the fly has only one α and two β-genes: the type of spectrin thus depends on which b chain is used.
Our goal is to understand how different spectrin isoforms integrate so many interactions in the domain with which each is associated, and how this integration contributes to overall cellular function and morphogenesis.
Key results behind our current thinking
1. Spectrin is NOT required for apicobasal polarity
By the late 1980’s hypotheses on the creation of an apicobasal axis in epithelial cells were starting to emerge from data on sequences directing the differential delivery of proteins. It was also a period of discovery in the spectrin field, which had brought the realization that spectrin isoforms were strongly polarized in a mutually exclusive fashion, and that they could stabilize several integral membrane proteins at the plasma membrane (it was presumed by anchoring –see 3 below). James Nelson’s lab put these three elements together to suggest that differential anchoring by spectrin isoforms might be central to the creation and stabilization of the apicobasal axis: If you randomly delivered an integral membrane protein, but it was only stably anchored by one spectrin isoform in one domain, the SBMS became a sorting machine.
As a postdoc in Dan Kiehart’s lab, I started working on βH-spectrin laying the groundwork for my lab to do the first test of the Nelson hypothesis – It was not true! While individual proteins might behave this way, we found Instead a surprising (at the time) cell adhesion defect in thata the zonula adherens was disrupted, but that this was NOT associated with a loss of apicobasal polarity. At Penn State, we followed up with another paper in collaboration with the LeBivic lab that demonstrated that yes, βH-spectrin was associated with the apical determinant Crumbs, but not in the polarity pathway per se. These are both very significant results, which led to a sea change our view of the non-erythroid spectrins.
Zarnescu, D.C. and Thomas, C.M. 1999. Apical spectrin is essential for epithelial morphogenesis but not apico-basal polarity in Drosophila. J. Cell Biol. 146;1075-1086. PMID: 10477760
Médina, E., Williams, J.A., Klipfell, E.A., Zarnescu, D.C., Thomas, C.M., Le Bivic, A. 2002. Crumbs interacts Moesin and βHeavy-Spectrin in the apical membrane skeleton of Drosophila J. Cell Biol. 158 941-951. PMID: 2213838
2. Spectrin has a prominent role in protein trafficking
So, just what is spectrin doing if it isn’t part of apicobasal axis formation? In 2000 Pinder and Baines had proposed that spectrin networks are ‘protein accumulators’: That is membrane proteins are delivered, then retained by anchoring to the network. Only when released could they be endocytosed. Our most recent papers paint a different picture. We had a hint that βH-spectrin had roles in the endosomal system when we discovered that an apical V‑type H+ATPase was delivered apically, but was lost to the lysosome in the absence of βH. A key epistasis experiment placed the requirement for βH-spectrin downstream of Dynamin (i.e. post-internalization) in stabilizing the pump. When we subsequently sought binding partners for βH-spectrin, we identified the peripheral endomembrane protein Annexin B9 as a partner. Reduction in Annexin B9 causes βH-spectrin to become trapped on mid- to late-endosmal compartments, and generates a traffic jam in the endosomal pathway before the multivesicular body. This and further evidence point towards the hypothesis that spectrin remains associated with internalizing vesicles and endosomes, and that it promotes recycling back to the plasma membrane. This is corroborated by a very similar phenotype created by reduction in a protein phosphatase that is also bound to βH-spectrin (in prep), and observations from another lab indicating that βH-spectrin binds to patronin in the exocytic pathway.
This has led us to propose a big change in our perspective on spectrin-based membrane protein stabilization that we call dynamic protein maintenance – we have proposed that spectrin forms a metazoan overlay on the core endosomal processes to promote rapid recycling, and that this works to maintain protein pool sizes at the plasma membrane. This does not deny the existence of non-erythroid networks, nor that some proteins are anchored, but it extends the influence of spectrin to unanchored proteins too (since attachment is to the vesicle).
Phillips M. and Thomas, C. M. 2006. Brush border spectrin is required for the early endosome recycling pathway in Drosophila. J. Cell Sci. 119;1361-1370. PMID: 16537648
Lee, H. G., Zarnescu, D. C., MacIver, B. and Thomas, C. M. (2010). The cell adhesion molecule Roughest depends on βHeavy-spectrin during eye morphogenesis in Drosophila. J. Cell Sci. 123, 277-85. PMID: 20048344
Tjota, M, Lee, S-K., Wu, J., Williams, J.A., Khanna, M.R. Thomas, C. M. (2011). Annexin B9 binds to βH-spectrin and is required for multivesicular body function in Drosophila. J. Cell Sci. 124, 2914–2926. PMID: 21878499
3. Non-erythroid spectrin network formation is less important than expected
The red cell has given us the dominant paradigm of a spectrin network that supports and strengthens the membrane. There is evidence that some non-erythroid networks have a similar role in neurons and during fusion to form muscle syncytia. However, a dynamic picture of epithelial spectrin-networks has been emerging. For example, about half the spectrin rapidly turns over during muscle fusion. We explicitly tested the need for F-actin crosslinking by spectrin – it’s most fundamental activity – and found to our great surprise that the network-busting mutation α-specR22S (R28S in human erythroid α-spectrin) has little to no phenotype. This was backed up with solid biochemistry showing that the mutation had the same effect in the fly protein, and starkly contrasts with studies which show that the total removal of α-spectrin gives a lethal phenotype. We do not deny that networks form, but the genetics is telling us that network formation per se is not as important in the fly as it is in the red cell.
Khanna, M.R., Crilly, S., Bakerink, K.J., Harper S., Speicher, D.W. and Thomas, C. M. (2015) Spectrin tetramer formation is not required for viable development in Drosophila. J. Biol. Chem. 290;706–715. PMID: 25381248
Duan, R., Kim, J.H., Shilagardi, K., Schiffhauer, E., Son, S., Lee, D., Li, S., Thomas, C., Luo, T., Fletcher, D.A., Robinson, D.N., and Chen, E.H. (2018) Spectrin is a mechanoresponsive protein shaping the architecture of intercellular invasion. Nature Cell Biol. (2018) 20;688–698 PMID: 29802406
