Intracellular Dynamin Elastin-like Polypeptides Assemble into Rodlike, Spherical, and Reticular Dynasomes.

Dynamin (DNM) is a family of large GTPases possessing a unique mechanical ability to "pinch" off vesicles entering cells. DNM2 is the most ubiquitously expressed member of the DNM family. We developed a novel tool based on elastin-like polypeptide (ELP) technology to quickly, precisely, and reversibly modulate the structure of DNM2. ELPs are temperature-sensitive biopolymers that self-assemble into microdomains above sharp transition temperatures. When linked together, DNM2 and a temperature-sensitive ELP fusion organize into a range of distinct temperature-dependent structures above a sharp transition temperature, which were not observed with wild-type DNM2 or a temperature-insensitive ELP fusion control. The structures comprised three different morphologies, which were prevalent at different temperature ranges. The size of these structures was influenced by an inhibitor of the DNM2 GTPase activity, dynasore; furthermore, they appear to entrap co-expressed cytosolic ELPs. Having demonstrated an unexpected diversity of morphologically distinct structures, DNM2-ELP fusions may have applications in the exploration of dynamin-dependent biology.

Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides.

Elastin-like polypeptides (ELPs) are modular, stimuli-responsive materials that self-assemble into protein-rich microdomains in response to heating. By cloning ELPs to effector proteins, expressed intracellular fusions can even modulate cellular pathways. A critical step in engineering these fusions is to determine and control their intracellular phase transition temperature (Tt). To do so, this Method paper describes a simple live-cell imaging technique to estimate the Tt of non-fluorescent ELP fusion proteins by co-transfection with a fluorescent ELP marker. Intracellular microdomain formation can then be visualized in live cells through the co-assembly of the non-fluorescent and fluorescent ELP fusion proteins. If the two ELP fusions have different Tt, the intracellular ELP mixture phase separates at the temperature corresponding to the fusion with the lower Tt. In addition, co-assembled ELP microdomains often exhibit pronounced differences in size or number, compared to single transfected treatments. These features enable live-cell imaging experiments and image analysis to determine the intracellular Tt of a library of related ELP fusions. As a case study, we employ the recently reported Caveolin1-ELP library (CAV1-ELPs). In addition to providing a detailed protocol, we also report the development of a useful FIJI plugin named SIAL (Simple Image Analysis Library), which contains programs for image randomization and blinding, phenotype scoring, and ROI selection. These tasks are important parts of the protocol detailed here and are also commonly employed in other image analysis workflows.

Caveolin elastin-like polypeptide fusions mediate temperature-dependent assembly of caveolar microdomains.

Caveolae are membrane organelles formed by submicron invaginations in the plasma membrane, and are involved in mechanosensing, cell signaling, and endocytosis. Although implicated broadly in physiology and pathophysiology, better tools are required to elucidate the precise role of caveolar processes through selective activation and inactivation of their trafficking. Our group recently reported that thermally-responsive elastin-like polypeptides (ELPs) can trigger formation of 'genetically engineered protein microdomains (GEPMs)' functionalized with either Clathrin-light chain or the epidermal growth factor receptor. This manuscript is the first report of this strategy to modulate caveolin-1 (CAV1). By attaching different ELP sequences to CAV1, mild heating can be used to self-assemble CAV1-ELP microdomains inside of cells. The temperature of self-assembly can be controlled by tuning the ELP sequence. The formation of CAV1-ELP microdomains internalizes Cholera Toxin Subunit B, a commonly used marker of caveolae mediated endocytosis. CAV1-ELPs also colocalize with Cavin 1, an essential component of functional caveolae biogenesis. With the emerging significance of caveolae in health and disease and the lack of specific probes to rapidly and reversibly affect caveolar function, CAV1-ELP microdomains are a new tool to rapidly probe caveolae associated processes in endocytosis, cell signaling, and mechanosensing.

A new temperature-dependent strategy to modulate the epidermal growth factor receptor.

The dynamic manipulation of kinases remains a major obstacle to unraveling cell-signaling networks responsible for the activation of biological systems. For example, epidermal growth factor (EGF) stimulates the epidermal growth factor receptor (EGFR/ErbB1); however, EGF also recruits other kinases (HER2/ErbB2) involved with various signaling pathways. To better study EGFR we report a new strategy to selectively activate receptor tyrosine kinases fused to elastin-like polypeptides (ELPs), which can be visualized inside mammalian cells using fixed and live-cell fluorescence microscopy. ELPs are high molecular weight polypeptides that phase separate abruptly upon heating. When an EGFR-ELP fusion is heated, it clusters, initiates receptor internalization, phosphorylates, initiates downstream kinase signaling, and undergoes retrograde transport towards the cell body. Unlike other strategies to block EGFR (small molecule inhibitors, RNAi, or transcriptional regulators), EGFR-ELP clustering can be specifically switched on or off within minutes. Live-cell imaging suggests that EGFR-ELPs assemble in most cells with only a 3 °C increase in temperature. This strategy was found reversible and able to dynamically control the downstream phosphorylation/activation of the ERK1/2 pathway. For the first time, this strategy enables the rational engineering of specific temperature-sensitive receptors that may have broad applications in the study and manipulation of biological processes.

Tunable assembly of protein-microdomains in living vertebrate embryos.

Subcellular events such as trafficking and signaling are regulated by self-assembled protein complexes inside the cell. The ability to rapidly and reversibly manipulate these protein complexes would likely enhance studies of their mechanisms and their roles in biological function and disease manifestation.[1, 2] This manuscript reports that thermally-responsive elastin-like polypeptides (ELPs) linked to fluorescent proteins can regulate the self-assembly and disassembly of protein microdomains within the individual cells of zebrafish embryos. By exploring a library of fluorescent ELP proteins, this reports demonstrates that ELPs can co-assemble different fluorescent proteins inside of embryos. By tuning ELP length and sequence, fluorescent protein microdomains can be assembled at different temperatures, in varying sizes, or for desired periods of time. For the first time in a multicellular living embryo, these studies demonstrate that temperature-mediated ELP assembly can reversibly manipulate assembly of subcellular protein complexes, which may have applications in the study and manipulation of in vivo biological functions.

Berunda Polypeptides: Multi-Headed Fusion Proteins Promote Subcutaneous Administration of Rapamycin to Breast Cancer In Vivo.

Recombinant Elastin-Like Polypeptides (ELPs) serve as attractive scaffolds for nanoformulations because they can be charge-neutral, water soluble, high molecular weight, monodisperse, biodegradable, and decorated with functional proteins. We recently reported that fusion of the FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) reduces rapamycin (Rapa) toxicity and enables intravenous (IV) therapy in both a xenograft breast cancer model and a murine autoimmune disease model. Rapa has poor solubility, which leads to variable oral bioavailability or drug precipitation following parenteral administration. While IV administration is routine during chemotherapy, cytostatic molecules like Rapa would require repeat administrations in clinical settings. To optimize FKBP/Rapa for subcutaneous (SC) administration, this manuscript expands upon first-generation FSI nanoparticles (Rh ~ 25 nm) and compares them with two second-generation carriers (FA and FAF) that: i) do not self-assemble; ii) retain a hydrodynamic radius (Rh ~ 7 nm) above the renal filtration cutoff; iii) increase tumor accumulation; and iv) have either one (FA) or two (FAF) drug-binding FKBP domains per ELP protein. Methods: The carriers were compared and evaluated for temperature-concentration phase behavior by UV-Vis spectrophotometry; equilibrium binding and thermodynamics by Isothermal Titration Calorimetry; drug retention and formulation stability by Dialysis and Dynamic Light Scattering; in vitro efficacy using a cell proliferation assay; in vivo efficacy in human MDA-MB-468 orthotopic breast cancer xenografts; downstream target inhibition using western blot; tissue histopathology; and bio-distribution via optical imaging in the orthotopic xenograft mouse model. Results: Named after the two-headed bird in Hindu mythology, the 'Berunda polypeptide' FAF with molecular weight of 97 kDa and particle size, Rh ~ 7 nm demonstrated polypeptide conformation of a soluble hydrated coiled polymer, retained formulation stability for one month post Rapa loading, eliminated toxicity observed with free Rapa after SC administration, suppressed tumor growth, decreased phosphorylation of a downstream target, and increased tumor accumulation in orthotopic breast tumor xenografts. Conclusion: This comprehensive manuscript demonstrates the versatility of recombinant protein-polymers to investigate drug carrier architectures. Furthermore, their facilitation of SC administration of poorly soluble drugs, like Rapa, may enable chronic self-administration in patients.