Eric Schiffhauer

Dual activation of CFTR and CLCN2 by lubiprostone in murine nasal epithelia.

Multiple sodium and chloride channels on the apical surface of nasal epithelial cells contribute to periciliary fluid homeostasis, a function that is disrupted in patients with cystic fibrosis (CF). Among these channels is the chloride channel CLCN2, which has been studied as a potential alternative chloride efflux pathway in the absence of CFTR. The object of the present study was to use the nasal potential difference test (NPD) to quantify CLCN2 function in an epithelial-directed TetOn CLCN2 transgenic mouse model (TGN-K18rtTA-hCLCN2) by using the putative CLCN2 pharmacological agonist lubiprostone and peptide inhibitor GaTx2. Lubiprostone significantly increased chloride transport in the CLCN2-overexpressing mice following activation of the transgene by doxycycline. This response to lubiprostone was significantly inhibited by GaTx2 after CLCN2 activation in TGN-CLCN2 mice. Cftr(-/-) and Clc2(-/-) mice showed hyperpolarization indicative of chloride efflux in response to lubiprostone, which was fully inhibited by GaTx2 and CFTR inhibitor 172 + GlyH-101, respectively. Our study reveals lubiprostone as a pharmacological activator of both CFTR and CLCN2. Overexpression and activation of CLCN2 leads to improved mouse NPD readings, suggesting it is available as an alternative pathway for epithelial chloride secretion in murine airways. The utilization of CLCN2 as an alternative chloride efflux channel could provide clinical benefit to patients with CF, especially if the pharmacological activator is administered as an aerosol.

Mechanochemical Signaling Directs Cell-Shape Change

For specialized cell function, as well as active cell behaviors such as division, migration, and tissue development, cells must undergo dynamic changes in shape. To complete these processes, cells integrate chemical and mechanical signals to direct force production. This mechanochemical integration allows for the rapid production and adaptation of leading-edge machinery in migrating cells, the invasion of one cell into another during cell-cell fusion, and the force-feedback loops that ensure robust cytokinesis. A quantitative understanding of cell mechanics coupled with protein dynamics has allowed us to account for furrow ingression during cytokinesis, a model cell-shape-change process. At the core of cell-shape changes is the ability of the cells machinery to sense mechanical forces and tune the force-generating machinery as needed. Force-sensitive cytoskeletal proteins, including myosin II motors and actin cross-linkers such as α-actinin and filamin, accumulate in response to internally generated and externally imposed mechanical stresses, endowing the cell with the ability to discern and respond to mechanical cues. The physical theory behind how these proteins display mechanosensitive accumulation has allowed us to predict paralog-specific behaviors of different cross-linking proteins and identify a zone of optimal actin-binding affinity that allows for mechanical stress-induced protein accumulation. These molecular mechanisms coupled with the mechanical feedback systems ensure robust shape changes, but if they go awry, they are poised to promote disease states such as cancer cell metastasis and loss of tissue integrity.

Interference with Ubiquitination in CFTR Modifies Stability of Core Glycosylated and Cell Surface Pools

ABSTRACT It is recognized that both wild-type and mutant CFTR proteins undergo ubiquitination at multiple lysines in the proteins and in one or more subcellular locations. We hypothesized that ubiquitin is added to specific sites in wild-type CFTR to stabilize it and at other sites to signal for proteolysis. Mass spectrometric analysis of wild-type CFTR identified ubiquitinated lysines 68, 710, 716, 1041, and 1080. We demonstrate that the ubiquitinated K710, K716, and K1041 residues stabilize wild-type CFTR, protecting it from proteolysis. The polyubiquitin linkage is predominantly K63. N-tail mutants, K14R and K68R, lead to increased mature band C CFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. The amount of CFTR in the K1041R mutant was drastically reduced and consisted of bands A/B, suggesting that the site in transmembrane 10 (TM10) was critical to further processing beyond the proteasome. The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition. Thus, ubiquitination at residue 1218 may destabilize wild-type CFTR in both the endoplasmic reticulum (ER) and recycling pools. Small molecules targeting the region of residue 1218 to block ubiquitination or to preserving structure at residues 710 to 716 might be protein sparing for some forms of cystic fibrosis.

Harnessing the adaptive potential of mechanoresponsive proteins to overwhelm pancreatic cancer dissemination and invasion

Metastatic disease is often characterized by altered cellular contractility and deformability, lending cells and groups of cells the flexibility to navigate through different microenvironments. This ability to change cell shape is driven in large part by the structural elements of the mechanobiome, which includes cytoskeletal proteins that sense and respond to mechanical stimuli. Here, we demonstrate that key mechanoresponsive proteins (those which accumulate in response to mechanical stress), specifically nonmuscle myosin IIA and IIC, α-actinin 4, and filamin B, are highly upregulated in pancreatic ductal adenocarcinoma cancer (PDAC) and in patient-derived pancreatic cancer cell lines. Their less responsive sister paralogs (myosin IIB, α-actinin 1, and filamin A) show a smaller dynamic range or disappear with PDAC progression. We demonstrate that these mechanoresponsive proteins directly impact cell mechanics using knock-down and overexpression cell lines. We further quantify the nonmuscle myosin II family members in patient-derived cell lines and identify a role for myosin IIC in the formation of transverse actin arcs in single cells and cortical actin belts in tissue spheroids. We harness the upregulation of myosin IIC and its impact of cytoskeletal architecture through the use of the mechanical modulator 4-hydroxyacetophenone (4-HAP), which increases myosin IIC assembly and stiffens cells. Here, 4-HAP decreases dissemination, induces cortical actin belts, and slows retrograde actin flow in spheroids. Finally, mice having undergone hemi-splenectomies with PDAC cells and then treated with 4-HAP have a reduction in liver metastases. Thus, increasing the activity of these mechanoresponsive proteins (in this case, by increasing myosin IIC assembly) to overwhelm the ability of cells to polarize and invade may be an effective strategy to improve the five-year survival rate of pancreatic cancer patients, currently hovering around 6%.

Spectrin is a mechanoresponsive protein shaping fusogenic synapse architecture during myoblast fusion

Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage. Here, we report that α/βH-spectrin (βH is also called karst) dynamically accumulates and dissolves at the fusogenic synapse between fusing Drosophila muscle cells, where an attacking fusion partner invades its receiving partner with actin-propelled protrusions to promote cell fusion. Using genetics, cell biology, biophysics and mathematical modelling, we demonstrate that spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse. The transiently accumulated spectrin network functions as a cellular fence to restrict the diffusion of cell-adhesion molecules and a cellular sieve to constrict the invasive protrusions, thereby increasing the mechanical tension of the fusogenic synapse to promote cell membrane fusion. Our study reveals a function of spectrin as a mechanoresponsive protein and has general implications for understanding spectrin function in dynamic cellular processes.Duan et al. find that the membrane skeleton protein spectrin is required for myoblast fusion in Drosophila, accumulating in a mechanosensitive manner in the receiving partner during cell–cell fusion to modulate adhesion and protrusion events.

Spectrin is a mechanoresponsive protein shaping the architecture of intercellular invasion

Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage1-3. Here, we report that spectrin dynamically accumulates and dissolves at the fusogenic synapse, where an attacking fusion partner mechanically invades its receiving partner with actin-propelled protrusions to promote cell-cell fusion4-7. Using genetics, cell biology, biophysics and mathematical modeling, we demonstrate that unlike myosin II that responds to dilation deformation, spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse. The accumulated spectrin forms an uneven network, which functions as a “sieve” to constrict the invasive fingerlike protrusions, thus putting the fusogenic synapse under high mechanical tension to promote cell membrane fusion. Taken together, our study has revealed a previously unrecognized function of spectrin as a dynamic mechanoresponsive protein that shapes the architecture of intercellular invasion. These findings have general implications for understanding spectrin function in other dynamic cellular processes beyond cell-cell fusion.

Parallel Compression Is a Fast Low-Cost Assay for the High-Throughput Screening of Mechanosensory Cytoskeletal Proteins in Cells

Cellular mechanosensing is critical for many biological processes, including cell differentiation, proliferation, migration, and tissue morphogenesis. The actin cytoskeletal proteins play important roles in cellular mechanosensing. Many techniques have been used to investigate the mechanosensory behaviors of these proteins. However, a fast, low-cost assay for the quantitative characterization of these proteins is still lacking. Here, we demonstrate that compression assay using agarose overlay is suitable for the high throughput screening of mechanosensory proteins in live cells while requiring minimal experimental setup. We used several well-studied myosin II mutants to assess the compression assay. On the basis of elasticity theories, we simulated the mechanosensory accumulation of myosin IIs and quantitatively reproduced the experimentally observed protein dynamics. Combining the compression assay with confocal microscopy, we monitored the polarization of myosin II oligomers at the subcellular level. The polarization was dependent on the ratio of the two principal strains of the cellular deformations. Finally, we demonstrated that this technique could be used on the investigation of other mechanosensory proteins.

Abstract 3811: The mechanobiome of pancreatic ductal adenocarcinoma: a new, targetable drug space

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality, with 37,000 people dying annually in the US. Existing strategies for treating cancer primarily mainly focus on inhibiting cell growth through specific genetic pathways, which typically either fail to completely abolish the disease or which lead to compensatory regulatory changes, and hence, drug resistance. Targeting cell mechanics remains an under-used approach for drug development. The direct driver of cell shape change events intrinsic to cellular functions, such migration and invasion, is the mechanobiome - a collection of cytoskeletal proteins which are the final determinants of a cell9s mechanical attributes and which lie downstream of KRAS and other regulatory molecules. Targeting, and ultimately, inhibiting these processes is less likely to be subject to compensatory regulation by cancer cells. We determined via western blot analysis and immunohistochemistry of patient-derived samples that key players involved in mechanosensation-myosin IIA, IIC, α-actin-4, and filamin B -show increased expression in cancerous ductal epithelial over normal tissue, while non-mechanosensory, or variable mechanosensory, paralogs (myosin IIB, α-actin-1, and filamin A) show decreased expression. This upregulation of highly mechanosensory proteins has initiated an investigation into the necessity and sufficiency of the myosin II paralogs in PDAC metastasis through overexpression and knockdown of expression, coupled with mechanical assays. In addition to resolving the mechanobiome of PDAC, we have previously shown that targeting of myosin IIC by 4-hydroxyacetophenone affects PDAC mechanics. We are testing the in vivo efficacy of 4-HAP by conducting a murine multi-arm study of metastatically human derived pancreatic cancer cells. Preliminary results suggest a protective effect against the metastasis of human pancreatic cancer cells among mice treated with 4-HAP every other day. Citation Format: Alexandra Surcel, Qingfeng Zhu, Eric Schiffhauer, Robert A. Anders, Douglas N. Robinson. The mechanobiome of pancreatic ductal adenocarcinoma: a new, targetable drug space. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3811.