Engineering Systems to Study the Mechanics of Cilia- and Airflow-mediated Mucus Clearance

The pulmonary airway surface liquid layer is comprised of two components: 1) inhaled pathogens that are stuck to a mucus layer and 2) a periciliary layer (PCL) that provides an environment for mucociliary clearance (MCC) out of the lungs. The mechanisms of how the beating of cilia from adjacent ciliated cells is coordinated are poorly understood. In Chapter 2, the perfusion of fluid flow along the apical surface of airway cells was hypothesized to yield airway cultures that transported mucus uni-directionally. To test this hypothesis, perfusion protocols were performed during ciliogenesis and post-ciliation phase of in vitro human bronchial epithelial (HBE) airway models. The length of exposure of fluid shear stress on the apical surface of airway epithelial cultures yielded transient or permanent unidirectional mucus transport in the direction of fluid flow cue. These characteristics matched in vivo biology and remained unseen in standard tissue culturing protocols. In addition to MCC, two other modes of mucus clearance have been studied namely cough clearance (CC) and proposed here, a third mechanism: cilia-independent "gas-liquid transport" (GLT). In Chapter 3, a system was engineered to deliver laminar, humidified airflow across the surface of HBE cultures, which emulated peak expiratory flow rates associated with exhalation. In the GLT models, three conditions of mucosal hydration were tested to represent a variety of clearance models between health and disease: from well-hydrated, normal-like mucus, in situ mucus, to dehydrated mucus, which represented severe Cystic Fibrosis (CF) mucus. At healthy mucus concentrations (2-4%), GLT rate was much faster at clearing mucus than MCC. In contrast, under conditions of severe dehydration, CF-like, GLT failed to produce significant mucus transport, as observed with MCC. In Chapter 4, the effect of mucus clearance with air velocities associated with cough was investigated and captured using high-speed photography. CC was also observed to decrease as mucus concentration increased. Together, the methods developed in this dissertation will help researchers to culture HBE cells with transport characteristics similar to in vivo behavior and help clinicians to better evaluate drug therapeutics on airway clearance for treating muco-obstructive diseases like CF.Doctor of Philosoph

Interfacial binding and aggregation of lamin A tail domains associated with Hutchinson–Gilford progeria syndrome

Hutchinson–Gilford progeria syndrome is a premature aging disorder associated with the expression of ∆50 lamin A (∆50LA), a mutant form of the nuclear structural protein lamin A (LA). ∆50LA is missing 50 amino acids from the tail domain and retains a C-terminal farnesyl group that is cleaved from the wild-type LA. Many of the cellular pathologies of HGPS are thought to be a consequence of protein–membrane association mediated by the retained farnesyl group. To better characterize the protein–membrane interface, we quantified binding of purified recombinant ∆50LA tail domain (∆50LA-TD) to tethered bilayer membranes composed of phosphatidylserine and phosphocholine using surface plasmon resonance. Farnesylated ∆50LA-TD binds to the membrane interface only in the presence of Ca[superscript 2 +] or Mg[superscript 2 +] at physiological ionic strength. At extremely low ionic strength, both the farnesylated and non-farnesylated forms of ∆50LA-TD bind to the membrane surface in amounts that exceed those expected for a densely packed protein monolayer. Interestingly, the wild-type LA-TD with no farnesylation also associates with membranes at low ionic strength but forms only a single layer. We suggest that electrostatic interactions are mediated by charge clusters with a net positive charge that we calculate on the surface of the LA-TDs. These studies suggest that the accumulation of ∆50LA at the inner nuclear membrane observed in cells is due to a combination of aggregation and membrane association rather than simple membrane binding; electrostatics plays an important role in mediating this association.National Institute of General Medical Sciences (U.S.) (1R01-GM101647)United States. Office of Naval Research. Presidential Early Career Award for Scientists and Engineers (N000141010562)National Institutes of Health (U.S.) (U01 EB014976

Membrane Association of the PTEN Tumor Suppressor: Molecular Details of the Protein-Membrane Complex from SPR Binding Studies and Neutron Reflection

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P2 were determined to be Kd∼12 µM and 0.4 µM, respectively, and Kd∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P2 and an increased apparent affinity to PI(3,4,5)P3, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P2 and no synergy in its binding with PS and PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN

Structure and Kinetics of the PTEN Tumor Suppressor: Investigation of Solution and Membrane-Associated States

<p>Cancer is an umbrella term for a class of diseases all characterized by uncontrolled cell growth. In 2008, the average person had a 20% probability of being diagnosed with cancer before the age of 75 and a 11% chance of dying from it [1]. There are two categories of genes whose alteration can result in cancer: 1) oncogenes that promote cell growth and 2) tumor-suppressor genes that inhibit cell division and survival. PTEN is the second-most frequently mutated gene in human cancer [2] after p53, which in turn is mutated in half of all tumors [3]. While PTEN has a protein phosphatase capacity, it fulfills its cancer protective role through its ability to dephosphorylate the lipid PI(3,4,5)P₃, which in turn shuts down the PI3K/Akt signaling pathway thereby downregulating cell growth. Despite its critical role in preventing aberrant cell proliferation, there is no structural information available on membrane-bound PTEN. The crystal structure of a highly truncated membrane-free PTEN mutant was determined [4], but the deleted N-terminal and C-terminal tails have postulated membrane-association and regulatory roles, respectively.</p> <p>Since PTEN’s regulatory function has been postulated to be membranemediated, it is crucial to identify the binding mechanism and the contribution of various lipid species to the overall kinetics. In this thesis, we first describe a novel biomimetic construct called a tethered bilayer lipid membrane (tBLM) which allows for the simultaneous characterization of the bilayer by multiple techniques while allowing for an exquisite control of lipid composition. To validate the biological relevance of tBLMs, we visualized their optical homogeneity using fluorescence microscopy (FM), quantified the defect-density using electrochemical impedance spectroscopy (EIS) and the lipid diffusivity using two-photon fluorescence correlation spectroscopy (2P-FCS). We formulated a protocol that allowed for the preparation of defect-free planar bilayers which were able to reproduce the fluidity of free-standing membranes (such as lipid vesicles) without compromising on long-term stability (the issue with black lipid membranes) while decoupling the proximal (to the substrate) leaflet from the distal leaflet (the issue with solid-supported lipid bilayers).</p> <p>We then proceeded to quantify the binding affinities of wt PTEN, an autismrelated mutant H93R PTEN, a Cowden syndrome-related mutant C124S PTEN and the truncated crystal structure PTEN mutant to the anionic lipids phosphatidylserine (PS), phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P₂] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P₃] individually, as well as to membranes composed of biologically-relevant mixtures of these lipids. We show that the membrane-association of PTEN is very sensitive to lipid composition. It had earlier been determined that PTEN has a very weak affinity for the zwitterionic phosphatidylcholine (PC) lipids [Kd > 500 μM] [5]. wt PTEN is initially attracted to the membrane through interactions with PS lipids [Kd=22.0±0.5 μM; Bmax=155±3 ng/cm2]. This is then followed by specific association with PI(4,5)P2 lipids [Kd=0.4±0.1 μM; Bmax=23±1 ng/cm2] which allows for the efficient dephosphorylation of the PI(3,4,5)P3 catalytic substrate [Kd=2.4±0.2 μM]. We also observed that wt PTEN shows an order of magnitude stronger affinity to membranes containing both PS and PI(4,5)P2 [Kd=0.04±0.01 μM] indicating a cooperative binding effect. The H93R PTEN mutation is spatially separated from the PI(4,5)P2-binding module (PBM), the CBR3 PS-binding motif in the C2 domain as well as the active site. Yet, it exhibits a 4-fold strengthened affinity to PS-containing membranes [Kd(H93R)=3.1±0.3 μM], a 3-fold weakened affinity to PI(4,5)P2-containing membranes [Kd(H93R)=1.3±0.2 μM] and a 75% reduced phosphatase activity [6] with respect to the wt. The catalytically inactive C124S mutation allows us to show that PTEN’s association with PI(4,5)P2 and PI(3,4,5)P3 is independent and non-competitive [Kd(PI(4,5)P2) = 0.32±0.03 μM; Kd(PI(3,4,5)P3)=0.12±0.03 μM; [Kd(PI(4,5)P2:PI(3,4,5)P3=1:1) = 0.13±0.01 μM], indicating distinct binding sites. The truncated PTEN mutant has a stronger association to PS lipids [2.5≤Kd(truncated)≤4.9 μM] compared to wt PTEN due to the increase in net charge of the protein by +14 as a result of the deletions and the loss of any unfavorable interactions between the tail and the body of the protein. We also observe a two-fold decreased affinity to PI(4,5)P2-bearing membranes compared to wt PTEN [Kd(truncated)=0.77±0.07 μM], likely due to the absence of six residues from the PI(4,5)P2 binding module (PBM).</p> <p>The SPR binding measurements serve a dual purpose of quantifying PTEN’s membrane association as well as identifying suitable conditions for performing neutron reflectivity (NR) measurements to determine the structure of membrane-bound PTEN. We studied four systems: H93R PTEN bound to a PS-bearing membrane, wt bound to a PS-bearing membrane, wt PTEN bound to a PS+PI(4,5)P2-bearing membrane and the truncated PTEN mutant bound to a PS+PI(4,5)P2-bearing membrane. The NR data was fit using the conventional slab/box model [7] as well as the new continuous distribution model that was recently developed by our group [8]. All four protein neutron scattering length density (nSLD) profiles are distinct, implying unique membrane-bound states. Both wt PTEN bound states show a 60 ˚A extension of the protein along the bilayer normal while the H93R bound state is more compact at an extension of just 45 ˚A. We suggest that this is primarily due to the C-terminal tail being located distal to the membrane for the wt PTEN, unlike for H93R PTEN, although the H93R point mutation could also result in a conformational change in the core domains of the protein. In all cases, there is minimal penetration of the protein into the lipid headgroups indicating an interfacial association of PTEN with the lipid bilayer</p> <p>We estimated the orientation of membrane-bound PTEN using the SASSIE [9] conformational generator in combination with Euler angle rotational analysis. Assuming that the core domains of the protein are unchanged from the crystal structure, wt PTEN binds to PS at an angle given by (θ, ϕ) = (30◦, 30◦) while wt PTEN binds to membranes containing both PS and PI(4,5)P2 at an angle given by (θ, ϕ) = (10◦, 300◦) where (θ, ϕ) = (0◦, 0◦) corresponds to the proposed membrane binding orientation of the protein, as predicted by the crystal structure [4]. This analysis fails when applied to the H93R PTEN NR data, likely indicating a deviation in the secondary structure of the mutant from the crystal structure.</p> <p>Finally, we performed complementary all-atom molecular dynamic (MD) simulations which allowed us to study the molecular-level details of PTEN’s equilibrium conformation(s), both in solution as well as in a membrane-bound state, while using the experimental results as a source of validation. The association of PTEN with a PS-bearing membrane results in a conformational change of the protein which provides the active site with easier access to the PI(3,4,5)P3 catalytic substrate. The C-terminal tail of membrane-bound PTEN is in a relatively compact conformation and is located distal to the membrane, making minimal contacts with the body of the protein, as suggested by the NR data. However, the tail is extended in solution, allowing it to associate with the CBR3 PS-binding motif of the C2 domain, thereby obstructing membrane association. While this is only one of the conformations that the tail can adopt, the PEST phosphorylation sites on the C-terminal tail are spatially adjacent to Lysines on the C2 domain. Consequently, multiple phosphorylations of the C-terminal tail could lock the protein in a ‘closed’ state where the tail interacts with the PS-binding sites, thereby excluding the possibility of membrane-association [10]. This implies the phosphorylation of the C-terminal tail is a plausible mechanism for PTEN regulation.</p> <p>In combination, these results from a broad spectrum of investigations provide an entirely new perspective on the activation and regulation of the PTEN tumor suppressor. The detailed molecular picture that arises is urgently needed to help define future research investigation into PTEN’s tumor suppressor role and aid in the search for pharmaceutical targets to counteract the adverse impact of PTEN mutations. They also provide a reference structure for a lipid phosphatase in its active state on a thermally disordered, in-plane fluid membrane. We showcase the ability of seemingly innocuous point mutations to disrupt the membrane-association process through a combination of altered interactions and conformational changes. Our data supports a regulatory role for the disordered C-terminal tail, based on its ability to defy the structure-function paradigm by interfering with PTEN’s ability to bind to the lipid membrane, thereby reducing its catalytic activity.</p