Alexandra Surcel

14-3-3 Coordinates Microtubules, Rac, and Myosin II to Control Cell Mechanics and Cytokinesis

BACKGROUND During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system. RESULTS To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, we used chemical genetics in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. We identified 14-3-3 and found that it is enriched in the cortex, helps maintain steady-state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain, and is needed to promote myosin II bipolar thick filament remodeling. CONCLUSIONS 14-3-3 connects microtubules, Rac, and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Furthermore, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament remodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes-cytokinesis cell shape change and cell mechanics.

Cytokinesis through biochemical-mechanical feedback loops.

Cytokinesis is emerging as a control system defined by interacting biochemical and mechanical modules, which form a system of feedback loops. This integrated system accounts for the regulation and kinetics of cytokinesis furrowing and demonstrates that cytokinesis is a whole-cell process in which the global and equatorial cortices and cytoplasm are active players in the system. Though originally defined in Dictyostelium, features of the control system are recognizable in other organisms, suggesting a universal mechanism for cytokinesis regulation and contractility.

Pharmacological activation of myosin II paralogs to correct cell mechanics defects

Significance Despite the integral role of cell mechanics, efforts to target mechanics for drug development have lagged. Here, we present an approach to identifying small molecules capable of modulating mechanics. We characterize 4-hydroxyacetophenone (4-HAP), isolated as a breakdown product of a hit from our pilot screen of over 22,000 compounds. We show that 4-HAP specifically alters the localization of the mechanoenzyme myosin II, increasing the stiffness of cells. The effect of 4-HAP on myosin II, whose specificity we have defined, occurs across phylogeny. In particular, we have demonstrated 4-HAP’s ability to convert the mechanical profile of metastasis-derived pancreatic cancer cells toward a normal WT-like state. Invasion and migration of these cells, which are hallmarks of the invasive capacity of malignant lesions, are decreased by 4-HAP. Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior.

Entosis Is Induced by Glucose Starvation

SUMMARY Entosis is a mechanism of cell death that involves neighbor cell ingestion. This process occurs in cancers and promotes a form of cell competition, where winner cells engulf and kill losers. Entosis is driven by a mechanical differential that allows softer cells to eliminate stiffer cells. While this process can be induced by matrix detachment, whether other stressors can activate entosis is unknown. Here, we find that entosis is induced in adherent cells by glucose withdrawal. Glucose withdrawal leads to a bimodal distribution of cells based on their deformability, where stiffer cells appear in a manner requiring the energy-sensing AMP-activated protein kinase (AMPK). We show that loser cells with high levels of AMPK activity are eliminated by winners through entosis, which supports winner cell proliferation under nutrient-deprived conditions. Our findings demonstrate that entosis serves as a cellular response to metabolic stress that enables nutrient recovery through neighbor cell ingestion.

7.5 Understanding How Dividing Cells Change Shape

Cytokinesis is an essential cellular process with significant developmental and medical implications. Fundamentally mechanical, this geometrically simple cell shape change encompasses nearly all cellular processes. Particularly featured are cytoskeletal mechanics, molecular motor mechanochemistry, fluid dynamics, and cellular physiology, all of which are carried out by genetically encoded biomolecules. This chapter presents the current understanding of how these processes and features contribute to the physical aspects of cytokinesis. The chapter is rounded out with a synthesis of the processes into what is emerging as an integrated control system characterized by mechanical and biochemical feedback loops.

Yes-Associated Protein impacts adherens junction assembly through regulating actin cytoskeleton organization

The Hippo pathway effector Yes-associated protein (YAP) regulates liver size by promoting cell proliferation and inhibiting apoptosis. However, recent in vivo studies suggest that YAP has important cellular functions other than controlling proliferation and apoptosis. Transgenic YAP expression in mouse hepatocytes results in severe jaundice. A possible explanation for the jaundice could be defects in adherens junctions that prevent bile from leaking into the blood stream. Indeed, immunostaining of E-cadherin and electron microscopic examination of bile canaliculi of Yap transgenic livers revealed abnormal adherens junction structures. Using primary hepatocytes from Yap transgenic livers and Yap knockout livers, we found that YAP antagonizes E-cadherin-mediated cell-cell junction assembly by regulating the cellular actin architecture, including its mechanical properties (elasticity and cortical tension). Mechanistically, we found that YAP promoted contractile actin structure formation by upregulating nonmuscle myosin light chain expression and cellular ATP generation. Thus, by modulating actomyosin organization, YAP may influence many actomyosin-dependent cellular characteristics, including adhesion, membrane protrusion, spreading, morphology, and cortical tension and elasticity, which in turn determine cell differentiation and tissue morphogenesis.

Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation

Genetic interaction analysis is used to identify new cytokinesis proteins involved in myosin II cleavage furrow accumulation and to demonstrate how different pathways collaborate to drive myosin II to the cleavage furrow. One of these proteins, RMD1, is required for myosin II cleavage furrow localization and acts in parallel with mechanical stress.

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%.

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.