Fluorescent protein development 1) We have launched a project to develop improved green photoactivatable fluorescent proteins (PAFPs) for Photoactivated Localization Microscopy (PALM). PALM is a super-resolution technique, which accesses information below the diffraction barrier of most optical microscopy techniques by localizing sparse subsets of molecules with high precision using two-dimensional Gaussian fits of their diffraction-limited fluorescence signals (spot size of 500 nm diameter). PAFPs make possible this technique by having molecules that are dark before a pulse of light turns on their fluorescence. This allows densely populated specimens to be imaged one or just a few molecules at a time, necessary for the molecular localization described above. Currently, PAFPs are available in essentially two colors, green and red. Red PAFPs have proven to be useful for PALM by providing low backgrounds and sufficient numbers of photons capable of <25 nm uncertainty in localization. Green PAFPs suffer from low photons and high backgrounds, which limit precise molecular localization. We are currently working on developing an improved green PAFP for PALM. Current status of the project is that variants of PAGFP have been developed with low background, but at the expense of photoactivation contrast. Current efforts are aimed at reengineering by site-directed mutagenesis high photoactivation contrast while maintaining the low background fluorescence. Yan Fu and Donald Johnson are working on this project. 2) One of the brightest conventional fluorescent proteins is PATagRFP. Working with Vladislav Verkhusha (Albert Einstein College of Medicine), we have developed a PATagRFP for use in a derivative molecular localization technique, sptPALM (single particle tracking PALM). This technique allows thousands of molecules to be track within the same cell providing readouts on diffusion of molecules within specific cell membrane regions. PATagRFP activates from a dark state to a brighter red molecule, whereas the mEos2 shifts from a green to red molecule. mEos2 is an excellent marker for sptPALM, but makes difficult 2-color experiments. Existing dark-to-red PAFPs lack the brightness and photostability of PATagRFP, so this improved variant has become the molecule of choice for 2-color sptPALM. 3) A project related to all PAFPs, whether current or under development, is the characterization of their blinking behaviors. Ideally, PAFPs would be turned on, fluoresce for a given period of time, photobleach, and never turn on again. However, common to almost all fluorophores, PAFPs sample dark states during which they do not produce photons. This is problematic since it is difficult to determine if a molecule has blinked or photobleached, and when it turns on once again it is difficult to determine if it is the same molecule or a different molecule. Simulations of blinking behavior indicate that these can dramatically affect molecule counting (density determinations) as well structural interpretations. We have devised assays and analyses to survey existing molecules in efforts to determine parameters that can correctly identify blinking molecules, combine their collected photons and correctly localize them only once. In addition, we are testing variants with mutations within and around the PAFP chromophores to determine which affect the blinking on times, blinking off times, and percentage of blinking molecules in a population. This project was initiated by a BESIP 2010 student, Caitlin Powell, and is currently pursued by Yan Fu. Collaborators on the project include the labs of Harald Hess (HHMI) and Jennifer Lippincott-Schwartz (NICHD). Cell biology projects 1) We are collaborating with Moshe Levi (UCHSC) on a project to image with PALM the localization of sodium phosphate transporters, NaPi-2a and NaPi-2c, in the apical brush border membrane (BMM) of opossum kidney (OK) cells. These molecules play key roles in renal proximal tubule inorganic phosphate (Pi) reabsorption and help maintain Pi homeostasis. These transporters respond to increases and decreases in dietary Pi by changing their abundance in the BMM via trafficking to and from the BMM and an intracellular compartment. The transporters are known to interact with a number of PDZ containing proteins, such as NHERF-1 and NHERF-3, and to be associated with microdomains enriched in cholesterol and glycosphingolipids. Our project will require multi-color PALM experiments on NaPi molecules and their interacting partners located in the convoluted apical membrane of OK cells under several conditions affecting molecule localization, molecule diffusion, and molecule clustering. Thus, we will need to localize the molecules in 3 dimensions within >1 m long membrane microvilli structures. These will be challenging experiments from both imaging and biology standpoints, but the obstacles are straightforward and we anticipate overcoming them as the project matures. Current status is developing and testing the PAFP tagged molecules expressed in COS 7 cells. These preparations include developing plasmids for expressing two molecules from the promoter to better control relative expression levels. This project currently being run by Yan Fu with assistance over the summer of 2010 from Monique McCants. 2) Our collaboration with Nihal Altan-Bonnet involves PALM imaging of plus strand RNA viral infection of cells. These viruses include picornaviruses, coronaviruses, and flaviviruses, which after infection of a cell dramatically remodel intracellular membranes into replication organelles on which RNA replication takes place. A number of intracellular proteins are hijacked during this process and recruited to the replication membranes. Our interest in this project is to image at super-resolution levels (<50nm) the localization of the numerous viral and host cell components on the replication organelles involved in replication at various times points post-infection. This project presents many of the same challenges as the NaPi project. This project is currently in the preliminary stages of observing host cell components in uninfected cells. 3) We collaborate with Wei-Shau Hu (NCI) on a project to localize the centers of mass of several proteins and RNAs within noninfectious Human Immunodeficiency Virus (HIV) particles. Genes for several critical components, Env, Vif, Vpu, and Vpr, have large deletions making the particles noninfectious. The lab of Wei-Shau Hu has developed several variants of these particles containing multiple color fluorescent protein tagged components. Since the particles (<100 nm diameter) are smaller than the resolution limit of conventional optics, the distribution of the different molecules appears as a diffraction-limited 500 nm spot. However, by two-dimensional Guassian fitting of the fluorescence distributions, we can provide precise localization of the average localization of the molecules. This differs from PALM since all molecules are imaged at once, but for the initial steps in this project we wish to simply define the relative positions of the particle components. This project is also in the preliminary stages and is under the guidance of George Patterson. Future plans include performing multi-color PALM experiments using PAFP tagged versions of the particle components.
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