Superresolution Imaging of the Enthesis Under Mechanical Load
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2126-Pos Board B856 Live Cell Imaging with Rab-Gtpases Elucidates Intracellular Pathways of RGD and iRGD Tagged Cationic Lipid-DNA Nanoparticles Ramsey N. Majzoub 1 , Kai K. Ewert 1 , Venkata R. Kotamraju 2,3 , Chia-Ling Chan 4,5 , Keng S. Liang 6,7 , Erkki Ruoslahti 3,8 , Cyrus R. Safinya 1 . 1 Department of Physics, Materials Department, University of California, Santa Barbara, Santa Barbara, CA, USA, 2 Cancer Research Center, Sanford- Burnham Medical Research Institute, La Jolla, CA, USA, 3 Center for Nanomedicine and Department of Cell, Molecular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA, 4 Department of Physics, University of California, Santa Barbara, Santa Barbara, CA, USA, 5 Institute of Physics, Academica Sinica, Taipei, Taiwan, 6 National Synchrotron Radiation Research Center, National Chiao-Tung University, Hsinchu, Taiwan, 7 Department of Electrophysics, National Chiao-Tung University, Hsinchu, Taiwan, 8 Cancer Center Research, Sanford Burnham Medical Research Institute, La Jolla, CA, USA. Steric stabilization of cationic lipid-DNA (CL-DNA) complexes is required for their use in vivo, but PEGylation (PEG; polyethylene glycol) of CL-DNA com- plexes reduces their efficacy as gene delivery vectors in vitro [1]. One approach to improving gene delivery with PEGylated CL-DNA nanoparticles is to cova- lently attach a targeting peptide at the distal end of PEG. We have developed PE- Gylated CL-DNA nanoparticles with an RGD or iRGD motif present at the distal end of PEG2000 and studied their efficacy and mechanism of entry in vitro. Studies have shown that nanoparticles exposing an RGD peptide are capable of targeting tumors in vivo via a specific interaction with the membrane bound receptor integrin. Recently, the next generation of RGD peptides, internalizing RGD (iRGD) has been shown to target and penetrate tumors in vivo [2]. Although neuropillin-1 is known to play a role in the tumor penetration proper- ties of iRGD, the intracellular mechanism which allows for tumor penetration is unknown. In order to investigate how iRGD promotes tumor penetration, we per- formed live cell imaging of fluorescently labeled RGD and iRGD-tagged CL- DNA nanoparticles using GFP-Rab-GTPases, an endosomal membrane bound protein which allows for spatio-temporal tracking of endosomal cargo. We present quantitative analysis using GFP-Rab-(5, 7 and 9) which label early and late endosomes and compare the colocalization of RGD and iRGD in these endosomes as a function of time. Finally, we investigated the gene delivery properties of RGD and iRGD-tagged lipid-DNA nanoparticles through the use of gene expression assays. A thorough understanding of the intracellular fate of CL-DNA nanoparticles will allow for optimization of gene delivery vec- tors. Funded by NIH-GM59288. [1] Chan, C.L. et al.; Biomaterials, 33, 4928-4935, 2012. [2] Sugahara, K.N. et al.; Cancer Cell, 16(6), 510-520, 2009. 2127-Pos Board B857 Biomimetic Light Harvesting in Nanoporous Metal Organic Materials Randy W. Larsen, Lukasz Wojtas, Chrsiti Whittington. Chemistry, University of South Florida, Tampa, FL, USA. Metal organic framework materials (MOFs) represent a class of solid state ma- terials with a number of properties advantageous for numerous applications ranging from gas storage and separation, heterogeneous catalysis, and controlled drug release to name only a few. Of the plethora of unexplored ap- plications of MOFs, frameworks that can facilitate biomimetic solar photo- chemistry are of significant interest. Solar photochemistry applications rely on the ability of the MOF material to undergo facile and directional photoin- duced electron transfer, much like biological light harvesting systems. One strategy is to utilize the nanoscale cavities within the MOF to encapsulate pho- toactive guests that can participate in directional photo-induced electron trans- fer reminiscent of biological electron transport chains. Here we discuss two such light harvesting systems. First, photoexcitation of a Zn(II)-trimesic acid based metal organic framework containing co-encapsulated Ru(II)tris(2,2’-bi- pyridine) (RuBpy) and Co(II)tris(2,2’-bipyridine) (CoBpy) results in inter- molecular electron transfer (ET) between the excited state of RuBpy ( 3 MLCT) and the ground state CoBpy The rate of inter-cavity ET, k ET , is found to be 3.7x10 6 s 1 . Using the semi-classical Marcus equation and the observed rate constant, it is determined that ET occurs between RuBpy and CoBpy com- plexes located in adjacent cavities (~19.6 A ˚ ). We have also previously demon- strated the ability to specifically encapsulate metall-porphyrins within the octahemioctahedral cavites of both Cu and Zn HKUST-1. Here the simulta- neous encapsulation of both Zn(II) tetrakis(tetra 4-sulphonatophenyl)porphyrin (Zn4SP) and Fe(III)tetrakis (tetra 4-sulphonatophenyl)porphyrin (Fe4SP) into a Zn(II)-HKUST-1 metal organic framework is demonstrated. Photo-excitation of the Zn4SP results in inter-molecular electron transfer (ET) between the encapsulated 3 Zn4SP and the Fe(III)4SP sites as evident by the reduction in 3 Zn4SP lifetime from 370 ms (k obs 2.7x10 3 s 1 ) to 83 ms (k obs = 1.2x10 4 s 1 ) in the presence of Fe4SP giving a k ET ~ 9.xx10 3 s 1 . 2128-Pos Board B858 Superresolution Imaging of the Enthesis Under Mechanical Load Heinrich Grabmayr 1 , Leone Rossetti 1 , Josef Stolberg-Stolberg 2 , Rainer Burgkart 2 , Andreas R. Bausch 1 . 1 Technische Universita ¨t Mu ¨nchen, Garching, Germany, 2 Technische Universita ¨t Mu ¨nchen, Mu ¨nchen, Germany. The enthesis is the anatomical site where bone and tendon join. At some loca- tions, such as the Achilles’ tendon insertion in the heel, forces transferred from muscle through tendon can exceed multiples of the body weight. Additional stress results from continuous changes in tendon orientation. Interfaces between materials with very different mechanical properties are known to be subject to early rupture caused by poor stress distribution, but the enthesis has an impressive durability. There currently only exists a limited understanding of the micromechanical and structural reasons behind this. We image the enthesis under mechanical stresses typical of physiological loads. To this end we designed a micromechanical load-chamber. This is combined with confocal and STED microscopies to investigate rat and pigsamples. Images taken with 60 nm resoltuion were stitched together to completely cover millimeter sized samples, allowing us to monitor the nanoscopic displacement of the collagen fibril and the strain fields on the scale of the whole sample, simultaneously. 2129-Pos Board B859 Investigation of Nanolipoprotein Particles Entrapped Within Nanoporous Silica: A Novel Platform for Immobilization of Integral Membrane Proteins Wade F. Zeno 1 , Marjorie L. Longo 1 , Subhash H. Risbud 2 , Matthew A. Coleman 3 . 1 Chemical Engineering, University of California, Davis, Davis, CA, USA, 2 Materials Science, University of California, Davis, Davis, CA, USA, 3 Lawrence Livermore National Laboratory, Livermore, CA, USA. Immobilization of integral membrane proteins (IMPs) in transparent, nanopo- rous silica gels has proven to be a challenge, as current and previous techniques utilize liposomes as biological membrane hosts. The instability of liposomes in nanoporous gels is attributed by their size (~150 nm) and altered structure and lipid dynamics upon entrapment within the nanometer scale pores (5-50 nm) of silica gel. This ultimately results in disruption of protein activity. We intend to overcome these barriers by using nanolipoprotein particles (NLPs) as bio- membrane hosts. NLPs are discoidal patches of lipid bilayer that are belted by amphiphilic scaffold proteins and have an average thickness of 5 nm, with diam- eters ranging from 10-15 nm. The IMP-NLP complexes are synthesized in a cell- free environment, which circumvents traditional protein reconstitution in mem- branes. Bacteriorhodopsin - a robust IMP protein that indicates its proper confor- mation via distinct purple coloration - will serve as a model IMP for this system. The spectral and physical properties of bacteriorhodopsin-NLPs entrapped within the gel are examined, as well as the phase behavior of the lipids within the NLP, to ensure proper functionality of the system. This bio-inorganic hybrid nanomaterial possesses a variety of viable applications. The success of this work could lead to the development of novel platforms in several areas, including high-throughput drug screening, chromatography, and biosensors. 2130-Pos Board B860 High Speed All Optical Logic Operations Utilizing the Protein Bacterio- rhodopsin La ´szlo ´ Fa ´bia ´n 1 , Anna Mathesz 1 , Sa ´ndor Valkai 1 , Daniel Alexandre 2 , Paulo V.S. Marques 2 , Pa ´l Ormos 1 , Elmar K. Wolff 3 , Andra ´s De ´r 1 . 1 Institute of Biophysics, Szeged, Hungary, 2 INESC-Porto, Porto, Portugal, 3 Institute for Applied Biotechnology and System Analysis, Witten, Germany. In data-processing applications requiring high speed and wide bandwidth, pho- tonic devices - where logic operations are processed on an all-optical basis - represent a promising alternative of their electronic counterparts. Besides in/ organic active optical crystals, dyes and polymers, molecules of biological origin with suitable nonlinear optical properties can also find applications in in- tegrated optical - biophotonic - devices. The principle of all-optical logical operations utilizing the unique nonlinear op- tical properties of a protein was demonstrated by a logic gate constructed from an integrated optical Mach-Zehnder interferometer as a passive structure, covered by a bacteriorhodopsin (bR) adlayer as the active element. Logical op- erations were based on a reversible change of the refractive index of the bR adlayer over one or both arms of the interferometer. Depending on the oper- ating point of the interferometer, we demonstrated binary and ternary logical modes of operation. Using an ultrafast transition of the bR photocycle (BR- K), we achieved high-speed (nanosecond) logical switching. This is the fastest operation of a protein-based integrated optical logic gate that has been demon- strated so far. The results are expected to have important implications for finding novel, alternative solutions in all-optical data processing research. Monday, February 17, 2014 421a
Transcript of Superresolution Imaging of the Enthesis Under Mechanical Load
Monday, February 17, 2014 421a
2126-Pos Board B856Live Cell Imaging with Rab-Gtpases Elucidates Intracellular Pathways ofRGD and iRGD Tagged Cationic Lipid-DNA NanoparticlesRamsey N. Majzoub1, Kai K. Ewert1, Venkata R. Kotamraju2,3,Chia-Ling Chan4,5, Keng S. Liang6,7, Erkki Ruoslahti3,8, Cyrus R. Safinya1.1Department of Physics, Materials Department, University of California,Santa Barbara, Santa Barbara, CA, USA, 2Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA, 3Center forNanomedicine and Department of Cell, Molecular and DevelopmentalBiology, University of California, Santa Barbara, Santa Barbara, CA, USA,4Department of Physics, University of California, Santa Barbara, SantaBarbara, CA, USA, 5Institute of Physics, Academica Sinica, Taipei, Taiwan,6National Synchrotron Radiation Research Center, National Chiao-TungUniversity, Hsinchu, Taiwan, 7Department of Electrophysics, NationalChiao-Tung University, Hsinchu, Taiwan, 8Cancer Center Research, SanfordBurnham Medical Research Institute, La Jolla, CA, USA.Steric stabilization of cationic lipid-DNA (CL-DNA) complexes is required fortheir use in vivo, but PEGylation (PEG; polyethylene glycol) of CL-DNA com-plexes reduces their efficacy as gene delivery vectors in vitro [1]. One approachto improving gene delivery with PEGylated CL-DNA nanoparticles is to cova-lently attach a targeting peptide at the distal end of PEG.We have developed PE-GylatedCL-DNAnanoparticles with anRGDor iRGDmotif present at the distalend of PEG2000 and studied their efficacy and mechanism of entry in vitro.Studies have shown that nanoparticles exposing an RGD peptide are capableof targeting tumors in vivo via a specific interaction with the membrane boundreceptor integrin. Recently, the next generation of RGD peptides, internalizingRGD (iRGD) has been shown to target and penetrate tumors in vivo [2].Although neuropillin-1 is known to play a role in the tumor penetration proper-ties of iRGD, the intracellular mechanism which allows for tumor penetration isunknown. In order to investigate how iRGDpromotes tumor penetration, we per-formed live cell imaging of fluorescently labeled RGD and iRGD-tagged CL-DNA nanoparticles using GFP-Rab-GTPases, an endosomal membrane boundprotein which allows for spatio-temporal tracking of endosomal cargo.We present quantitative analysis using GFP-Rab-(5, 7 and 9) which label earlyand late endosomes and compare the colocalization of RGD and iRGD in theseendosomes as a function of time. Finally, we investigated the gene deliveryproperties of RGD and iRGD-tagged lipid-DNA nanoparticles through theuse of gene expression assays. A thorough understanding of the intracellularfate of CL-DNA nanoparticles will allow for optimization of gene delivery vec-tors. Funded by NIH-GM59288.[1] Chan, C.L. et al.; Biomaterials, 33, 4928-4935, 2012.[2] Sugahara, K.N. et al.; Cancer Cell, 16(6), 510-520, 2009.
2127-Pos Board B857Biomimetic Light Harvesting in Nanoporous Metal Organic MaterialsRandy W. Larsen, Lukasz Wojtas, Chrsiti Whittington.Chemistry, University of South Florida, Tampa, FL, USA.Metal organic framework materials (MOFs) represent a class of solid state ma-terials with a number of properties advantageous for numerous applicationsranging from gas storage and separation, heterogeneous catalysis, andcontrolled drug release to name only a few. Of the plethora of unexplored ap-plications of MOFs, frameworks that can facilitate biomimetic solar photo-chemistry are of significant interest. Solar photochemistry applications relyon the ability of the MOF material to undergo facile and directional photoin-duced electron transfer, much like biological light harvesting systems. Onestrategy is to utilize the nanoscale cavities within the MOF to encapsulate pho-toactive guests that can participate in directional photo-induced electron trans-fer reminiscent of biological electron transport chains. Here we discuss twosuch light harvesting systems. First, photoexcitation of a Zn(II)-trimesic acidbased metal organic framework containing co-encapsulated Ru(II)tris(2,2’-bi-pyridine) (RuBpy) and Co(II)tris(2,2’-bipyridine) (CoBpy) results in inter-molecular electron transfer (ET) between the excited state of RuBpy(3MLCT) and the ground state CoBpy The rate of inter-cavity ET, kET, is foundto be 3.7x106 s�1. Using the semi-classical Marcus equation and the observedrate constant, it is determined that ET occurs between RuBpy and CoBpy com-plexes located in adjacent cavities (~19.6 A). We have also previously demon-strated the ability to specifically encapsulate metall-porphyrins within theoctahemioctahedral cavites of both Cu and Zn HKUST-1. Here the simulta-neous encapsulation of both Zn(II) tetrakis(tetra 4-sulphonatophenyl)porphyrin(Zn4SP) and Fe(III)tetrakis (tetra 4-sulphonatophenyl)porphyrin (Fe4SP) into aZn(II)-HKUST-1 metal organic framework is demonstrated. Photo-excitationof the Zn4SP results in inter-molecular electron transfer (ET) between theencapsulated 3Zn4SP and the Fe(III)4SP sites as evident by the reduction in3Zn4SP lifetime from 370 ms (kobs 2.7x10
3 s�1) to 83 ms (kobs = 1.2x104 s�1)in the presence of Fe4SP giving a kET ~ 9.xx103 s�1.
2128-Pos Board B858Superresolution Imaging of the Enthesis Under Mechanical LoadHeinrich Grabmayr1, Leone Rossetti1, Josef Stolberg-Stolberg2,Rainer Burgkart2, Andreas R. Bausch1.1Technische Universitat Munchen, Garching, Germany, 2TechnischeUniversitat Munchen, Munchen, Germany.The enthesis is the anatomical site where bone and tendon join. At some loca-tions, such as the Achilles’ tendon insertion in the heel, forces transferred frommuscle through tendon can exceed multiples of the body weight. Additionalstress results from continuous changes in tendon orientation.Interfaces between materials with very different mechanical properties areknown to be subject to early rupture caused by poor stress distribution, butthe enthesis has an impressive durability. There currently only exists a limitedunderstanding of the micromechanical and structural reasons behind this.We image the enthesis under mechanical stresses typical of physiological loads.To this endwedesigned amicromechanical load-chamber. This is combinedwithconfocal andSTEDmicroscopies to investigate rat andpig samples. Images takenwith 60nmresoltuionwere stitched together to completely covermillimeter sizedsamples, allowing us to monitor the nanoscopic displacement of the collagenfibril and the strain fields on the scale of the whole sample, simultaneously.
2129-Pos Board B859Investigation of Nanolipoprotein Particles Entrapped Within NanoporousSilica: A Novel Platform for Immobilization of Integral MembraneProteinsWade F. Zeno1, Marjorie L. Longo1, Subhash H. Risbud2,Matthew A. Coleman3.1Chemical Engineering, University of California, Davis, Davis, CA, USA,2Materials Science, University of California, Davis, Davis, CA, USA,3Lawrence Livermore National Laboratory, Livermore, CA, USA.Immobilization of integral membrane proteins (IMPs) in transparent, nanopo-rous silica gels has proven to be a challenge, as current and previous techniquesutilize liposomes as biological membrane hosts. The instability of liposomes innanoporous gels is attributed by their size (~150 nm) and altered structure andlipid dynamics upon entrapment within the nanometer scale pores (5-50 nm)of silica gel. This ultimately results in disruption of protein activity. We intendto overcome these barriers by using nanolipoprotein particles (NLPs) as bio-membrane hosts. NLPs are discoidal patches of lipid bilayer that are belted byamphiphilic scaffold proteins and have an average thickness of 5 nm, with diam-eters ranging from10-15 nm. The IMP-NLP complexes are synthesized in a cell-free environment, which circumvents traditional protein reconstitution in mem-branes. Bacteriorhodopsin - a robust IMPprotein that indicates its proper confor-mation via distinct purple coloration - will serve as a model IMP for this system.The spectral and physical properties of bacteriorhodopsin-NLPs entrappedwithin the gel are examined, as well as the phase behavior of the lipids withinthe NLP, to ensure proper functionality of the system. This bio-inorganic hybridnanomaterial possesses a variety of viable applications. The success of this workcould lead to the development of novel platforms in several areas, includinghigh-throughput drug screening, chromatography, and biosensors.
2130-Pos Board B860High Speed All Optical Logic Operations Utilizing the Protein Bacterio-rhodopsinLaszlo Fabian1, Anna Mathesz1, Sandor Valkai1, Daniel Alexandre2,Paulo V.S. Marques2, Pal Ormos1, Elmar K. Wolff3, Andras Der1.1Institute of Biophysics, Szeged, Hungary, 2INESC-Porto, Porto, Portugal,3Institute for Applied Biotechnology and System Analysis, Witten, Germany.In data-processing applications requiring high speed and wide bandwidth, pho-tonic devices - where logic operations are processed on an all-optical basis -represent a promising alternative of their electronic counterparts. Besides in/organic active optical crystals, dyes and polymers, molecules of biologicalorigin with suitable nonlinear optical properties can also find applications in in-tegrated optical - biophotonic - devices.The principle of all-optical logical operations utilizing the unique nonlinear op-tical properties of a protein was demonstrated by a logic gate constructed froman integrated optical Mach-Zehnder interferometer as a passive structure,covered by a bacteriorhodopsin (bR) adlayer as the active element. Logical op-erations were based on a reversible change of the refractive index of the bRadlayer over one or both arms of the interferometer. Depending on the oper-ating point of the interferometer, we demonstrated binary and ternary logicalmodes of operation. Using an ultrafast transition of the bR photocycle (BR-K), we achieved high-speed (nanosecond) logical switching. This is the fastestoperation of a protein-based integrated optical logic gate that has been demon-strated so far. The results are expected to have important implications forfinding novel, alternative solutions in all-optical data processing research.
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