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Lis1 has Two Distinct Modes of Regulating Dynein's Mechanochemical Cycle
Sunday, February 12, 2017 vesicles (MLVs). More recently, giant unilamellar vesicles (GUVs) have been used, which are excellent to study lipid phase separation, especially by fluorescence confocal microscopy, but do not easily lend themselves to calorimetry. However, the heat capacity of DPPC across the main phase transition is similar in GUVs and in large unilamellar vesicles (LUVs) but quite different from that in MLVs. Much of the attention in the thermodynamics of the phase transition in DPPC/cholesterol has been concerned with understanding the heat capacity in MLVs. Here we turn our attention to the DPPC/cholesterol binary system in LUVs, which we think is a much more relevant type of vesicle to understand the molecular interactions between DPPC and cholesterol. We compare the experimental heat capacity (melting) curves in LUVs with the results of Monte Carlo calculations using various models of the interaction between these lipids, including complex formation and simple pairwise interactions. This work has been supported by NSF grant CHE-1464769. 217-Plat Characterization of the Physiochemical Interactions between LNPs and the Endosomal Lipids: A Rational Design of Gene Delivery Systems Nandhitha Subramanian, Yoav Atsmon-Raz, Peter D. Tieleman. Department of Biological Sciences & Centre for Molecular Simualtion, University of Calgary, Calgary, AB, Canada. Lipid nanoparticles (LNPs) are a class of materials, with each interacting in a complex fashion to form specialized bio-containers carrying therapeutic drugs and strands of nucleic acids such as small interfering RNA (siRNA)1. LNPs are at the forefront of the rapidly developing field of nanotechnology with several potential applications in drug delivery, clinical medicine, and research. Despite holding a significant promise for reaching the goal of controlled and site specific drug delivery, the engineering of efficient LNPs is limited by the lack of knowledge about the structure of LNPs, the roles of their individual components and the physical interactions with other biomolecules2. Because of this, characteristics such as the encapsulation efficiency, polydispersity, and stability of LNPs are not predictable. One of the biggest problems of LNP gene therapy is the molecular details of the cellular processes that determine the efficiency of intracellular drug delivery is still unclear. Studies have shown that LNPs enter cells via the endocytic pathway and are engulfed by endosomes. The delivery of siRNA is substantially reduced, as ~70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes3. In this project, we used advanced molecular dynamics simulations to understand the molecular basis of the structure of LNPs based on their composition. Our simulations also provided the first detailed molecularlevel view of the interactions of the LNP components with endosomal lipids. Here, we also studied the change in interactions between the LNPs and endosomal lipids by varying ion concentration and altering pH. Results of our simulation will provide a route to the rational design of new LNPs that address safety concerns and ensure effective delivery to accelerate the translation of engineering lipid-based nanoparticles towards the clinic. References 1. Semple, S.C., et al. (2010) Rational design of cationic lipids for siRNA delivery. Nat.Biotechnol. 28,172-176 2. Huang, L. and Liu, Y. (2011) In vivo delivery of RNAi with lipid-based nanoparticles. Annu.Rev.Biomed. Eng. 13, 507-530 3. Sahay G., et al. (2013) Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat.Biotechnol. 31,653-658. 218-Plat Tuning Membrane Asymmetry: Controlled Uptake of Negatively Charged Lipids into the Outer Leaflet of Liposomes Marie Markones1, Carina Zorzin1, Louma Kalie1, Sebastian Fiedler2, Heiko Heerklotz2,3. 1 Institute for Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany, 2Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada, 3Institute for Pharmaceutical Sciences/BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany. The presence of negatively charged lipids in the outer leaflets of cellular membranes is a key component of many biological membranes. For example, eukaryotic organisms accumulate negatively charged lipids on their extracellular membrane leaflets during programmed cell death through apoptosis. As a general feature, the outer leaflets of bacterial cellular membranes exhibit high contents of anionic phosphatidylglycerol (PG). Recently, a cyclodextrin-based lipid exchange assay has been introduced that transports phospholipids from the outer leaflet of donor liposomes to that of acceptor liposomes, thereby generating model membranes of asymmetric lipid composition. In an effort to master and further develop the cyclodextrin method, we utilize the zeta potential of mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG)/1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes of symmetric and
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asymmetric composition to quantify the incorporation of negatively charged POPG into POPC liposomes. The adjustment of cyclodextrin and lipid concentrations below the maximum lipid-binding capacity of cyclodextrins allows a controlled POPG transfer without the need of donor liposomes. Furthermore, the zeta potential clearly differentiates between asymmetric and symmetric POPG incorporation, quantifies the degree of lipid asymmetry, and determines the total yield of lipid exchange. Our approach represents an easy to use method for the efficient incorporation of negatively charged lipids into the outer membrane leaflet of liposomes at physiologically relevant lipid contents. 219-Plat Investigating the Effects of the Membrane Dipole Field on the Structure and Function of a Model Membrane Protein Cari M. Anderson, Lauren J. Webb. Chemistry, University of Texas at Austin, Austin, TX, USA. Lipid bilayer membranes are composed of hundreds of lipids, sterols and proteins, which organize in to a heterogeneous, structural scaffold that controls critical elements of biological function. The interactions of all of these molecules are mostly non-covalent and electrostatic in nature. These interactions and the diversity of molecules, along with ordered water molecules at the lipid-water interface gives rise to a large electrostatic field that traverses the lipid bilayer. The whole field can be broken down in to three componentstransmembrane field, surface field and dipole field. We have extensively studied the membrane dipole field, which propagates from the interior of the lipid bilayer to the lipid head group-water interface, using vibrational Stark effect spectroscopy paired with molecular dynamics simulations. With our knowledge of how the dipole field can be altered due to a change in lipid bilayer composition, we are exploring what role this field plays in controlling the transport of ions through a membrane via transmembrane protein channels (TPCs). Gramicidin has been accepted as a good model for TPCs due to its ability to selectively transport ions with a þ1 charge through the pore it creates when in its channel conformation. Using this well characterized model TPC we hope to elucidate how the structure and function can be changed via noncovalent interactions with other lipids, water molecules and sterols, specifically arising from the membrane dipole field. Using VSE spectroscopy, we have been able to begin to measure how gramicidin alters the dipole field of a pure phospholipid bilayer in the form of small unilamellar vesicles (SUVs). We are studying how the transport of þ1 cations through the gramicidin channel and the structure of the TPC are affected by both small and large perturbations of the electrostatic field by intercalating sterols in to the lipid bilayer.
Platform: Kinesins and Dyneins 220-Plat Lis1 has Two Distinct Modes of Regulating Dynein’s Mechanochemical Cycle Michael A. Cianfrocco, Morgan E. DeSantis, Zaw M. Htet, Phuoc T. Tran, Andres E. Leschziner, Samara L. Reck-Peterson. Cellular and Molecular Medicine, University of California - San Diego, La Jolla, CA, USA. Cytoplasmic dynein-1 (‘‘dynein’’) is a minus-end-directed microtubule-based motor that couples ATP hydrolysis to force generation to move diverse cargos. Dynein is a single-chain AAAþ ATPase that contains 6 AAAþ domains, where AAA1-4 bind nucleotide and AAA1 drives the motor. Lis1 is a conserved and ubiquitous dynein regulator. Previously, we showed that Lis1 binds to dynein at AAA4 and causes dynein to slow down and remain attached to microtubules, even in the presence of ATP, which usually releases dynein from its track. Interestingly, while AAA3 is occupied by ADP when dynein is walking, either the absence of nucleotide or the presence of ATP lead to a motor that behaves like its Lis1-regulated state. This observation led us to hypothesize that Lis1 acts through AAA3. To test this we determined how the nucleotide state at AAA3 affects Lis1’s regulation of dynein. When AAA3 is nucleotide-free, Lis1 increases dynein’s microtubule binding affinity, as we had previously observed, Surprisingly, however, when AAA3 contains ATP, Lis1 has the opposite effect, leading to dynein’s detachment from microtubules. High-resolution cryo-electron microscopy structures of dynein-Lis1 complexes revealed the basis for these puzzling effects. While a single Lis1 beta propeller (Lis1 is a dimer) binds to dynein in the AAA3-(no nucleotide) state, a second Lis1 beta propeller is bound to the motor in the AAA3-ATP state. This novel second site is located on dynein’s coiled coil ‘‘stalk’’, which connects dynein’s motor domain to its microtubule binding domain. Importantly, the sequence of this site is conserved only in those dyneins that are regulated by Lis1. Our work revealed that Lis1 can act either as a microtubule anchor or a release factor for dynein, depending upon the nucleotide occupancy at AAA3. We propose a new model for how Lis1 serves as a dual regulator of dynein activity in cells.
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asymmetric composition to quantify the incorporation of negatively charged POPG into POPC liposomes. The adjustment of cyclodextrin and lipid concentrations below the maximum lipid-binding capacity of cyclodextrins allows a controlled POPG transfer without the need of donor liposomes. Furthermore, the zeta potential clearly differentiates between asymmetric and symmetric POPG incorporation, quantifies the degree of lipid asymmetry, and determines the total yield of lipid exchange. Our approach represents an easy to use method for the efficient incorporation of negatively charged lipids into the outer membrane leaflet of liposomes at physiologically relevant lipid contents. 219-Plat Investigating the Effects of the Membrane Dipole Field on the Structure and Function of a Model Membrane Protein Cari M. Anderson, Lauren J. Webb. Chemistry, University of Texas at Austin, Austin, TX, USA. Lipid bilayer membranes are composed of hundreds of lipids, sterols and proteins, which organize in to a heterogeneous, structural scaffold that controls critical elements of biological function. The interactions of all of these molecules are mostly non-covalent and electrostatic in nature. These interactions and the diversity of molecules, along with ordered water molecules at the lipid-water interface gives rise to a large electrostatic field that traverses the lipid bilayer. The whole field can be broken down in to three componentstransmembrane field, surface field and dipole field. We have extensively studied the membrane dipole field, which propagates from the interior of the lipid bilayer to the lipid head group-water interface, using vibrational Stark effect spectroscopy paired with molecular dynamics simulations. With our knowledge of how the dipole field can be altered due to a change in lipid bilayer composition, we are exploring what role this field plays in controlling the transport of ions through a membrane via transmembrane protein channels (TPCs). Gramicidin has been accepted as a good model for TPCs due to its ability to selectively transport ions with a þ1 charge through the pore it creates when in its channel conformation. Using this well characterized model TPC we hope to elucidate how the structure and function can be changed via noncovalent interactions with other lipids, water molecules and sterols, specifically arising from the membrane dipole field. Using VSE spectroscopy, we have been able to begin to measure how gramicidin alters the dipole field of a pure phospholipid bilayer in the form of small unilamellar vesicles (SUVs). We are studying how the transport of þ1 cations through the gramicidin channel and the structure of the TPC are affected by both small and large perturbations of the electrostatic field by intercalating sterols in to the lipid bilayer.
Platform: Kinesins and Dyneins 220-Plat Lis1 has Two Distinct Modes of Regulating Dynein’s Mechanochemical Cycle Michael A. Cianfrocco, Morgan E. DeSantis, Zaw M. Htet, Phuoc T. Tran, Andres E. Leschziner, Samara L. Reck-Peterson. Cellular and Molecular Medicine, University of California - San Diego, La Jolla, CA, USA. Cytoplasmic dynein-1 (‘‘dynein’’) is a minus-end-directed microtubule-based motor that couples ATP hydrolysis to force generation to move diverse cargos. Dynein is a single-chain AAAþ ATPase that contains 6 AAAþ domains, where AAA1-4 bind nucleotide and AAA1 drives the motor. Lis1 is a conserved and ubiquitous dynein regulator. Previously, we showed that Lis1 binds to dynein at AAA4 and causes dynein to slow down and remain attached to microtubules, even in the presence of ATP, which usually releases dynein from its track. Interestingly, while AAA3 is occupied by ADP when dynein is walking, either the absence of nucleotide or the presence of ATP lead to a motor that behaves like its Lis1-regulated state. This observation led us to hypothesize that Lis1 acts through AAA3. To test this we determined how the nucleotide state at AAA3 affects Lis1’s regulation of dynein. When AAA3 is nucleotide-free, Lis1 increases dynein’s microtubule binding affinity, as we had previously observed, Surprisingly, however, when AAA3 contains ATP, Lis1 has the opposite effect, leading to dynein’s detachment from microtubules. High-resolution cryo-electron microscopy structures of dynein-Lis1 complexes revealed the basis for these puzzling effects. While a single Lis1 beta propeller (Lis1 is a dimer) binds to dynein in the AAA3-(no nucleotide) state, a second Lis1 beta propeller is bound to the motor in the AAA3-ATP state. This novel second site is located on dynein’s coiled coil ‘‘stalk’’, which connects dynein’s motor domain to its microtubule binding domain. Importantly, the sequence of this site is conserved only in those dyneins that are regulated by Lis1. Our work revealed that Lis1 can act either as a microtubule anchor or a release factor for dynein, depending upon the nucleotide occupancy at AAA3. We propose a new model for how Lis1 serves as a dual regulator of dynein activity in cells.