Michelle K. Knowles

Sensing Muscle Ischemia: Coincident Detection of Acid and ATP via Interplay of Two Ion Channels

Ischemic pain--examples include the chest pain of a heart attack and the leg pain of a 30 s sprint--occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here, we show that another compound released from ischemic muscle, adenosine tri-phosphate (ATP), works together with acid by increasing the pH sensitivity of acid-sensing ion channel number 3 (ASIC3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis.

Syntaxin clusters assemble reversibly at sites of secretory granules in live cells

Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

Single secretory granules of live cells recruit syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in large copy numbers

Before secretory vesicles undergo exocytosis, they must recruit the proteins syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in the plasma membrane. GFP-labeled versions of both proteins cluster at sites where secretory granules have docked. Single-particle tracking shows that minority populations of both molecules are strongly hindered in their mobility, consistent with their confinement in nanodomains. We measured the fluorescence of granule-associated clusters, the fluorescence of single molecules, and the numbers of unlabeled syntaxin-1 and SNAP-25 molecules per cell. There was a more than 10-fold excess of SNAP-25 over syntaxin-1. Fifty to seventy copies each of syntaxin-1 and SNAP-25 molecules were associated with a single docked granule, many more than have been reported to be required for fusion.

Cytoskeletal-assisted dynamics of the mitochondrial reticulum in living cells

Subcellular organelle dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, and lead to complex multiexponential relaxations that occur over a wide range of spatial and temporal scales. Here we report spatio-temporal measurements of the fluctuations of the mitochondrial reticulum in osteosarcoma cells by using Fourier imaging correlation spectroscopy, over time and distance scales of 10−2 to 103 s and 0.5–2.5 μm. We show that the method allows a more complete description of mitochondrial dynamics, through the time- and length-scale-dependent collective diffusion coefficient D(k,τ), than available by other means. Addition of either nocodazole to disrupt microtubules or cytochalasin D to disassemble microfilaments simplifies the intermediate scattering function. When both drugs are used, the reticulum morphology of mitochondria is retained even though the cytoskeletal elements have been de-polymerized. The dynamics of the organelle are then primarily diffusive and can be modeled as a collection of friction points interconnected by elastic springs. This study quantitatively characterizes organelle dynamics in terms of collective cytoskeletal interactions in living cells.

A replicating module as the unit of mitochondrial structure and functioning

The mitochondrion within human cells in tissue culture is pleomorphic and highly dynamic. The organelle mass can exist as thousands of small ovoids or as one continuous reticulum. In either state, the mitochondrial mass is in constant thermal motion, as well as moving in approximately 0.8-microm jumps that are determined by, and related to, attachments with cytoskeletal elements. Many protein complexes, such as the pyruvate dehydrogenase (PDH) complex and DNA containing nucleoids, are dispersed through the mass and as though fixed by attachments to membranes, such that they can become distributed to all of the individual small ovoid mitochondria when the reticulum becomes fragmented. This leads us to propose that a replicating module is the repeating unit of mitochondrial structure. Studies to examine heterogeneity of functioning within the organelle mass are briefly reviewed.

C-reactive protein (CRP) aptamer binds to monomeric but not pentameric form of CRP

AbstractNative C-reactive protein (CRP) is composed of five identical subunits arranged in a pentameric structure (pCRP). Binding of pCRP to damaged cell membranes produces a second isoform, modified CRP, which has similar antigenicity to isolated monomeric subunits of CRP (mCRP). Emerging evidence indicates that modified CRP plays a role in inflammation and atherosclerosis, however, there are very few techniques that can distinguish the different isoforms of CRP. Here we show that an RNA aptamer binds specifically to mCRP and not to pCRP. Using this aptamer, we describe a simple, fast, and sensitive assay to detect nanomolar concentrations of mCRP using fluorescence anisotropy. In addition, we show that this aptamer can be used to detect mCRP in polyacrylamide gels and bound to a surface using total internal reflection fluorescence microscopy. The biological activity of the mCRP we prepared by heating pCRP with 0.1% sodium dodecyl sulfate was confirmed by observing binding to the complement protein, C1q. This probe provides an important tool for CRP research and has the potential to improve clinical diagnostics that predict risk for cardiovascular disease. FigureEvidence for mCRP selectivity of aptamer by gel electrophoresis, fluorescence anisotropy, and TIRF microscopy

Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature

The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and hexadecanoic acid (HDA), using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature. Both lipids studied contain 16 carbon, saturated tails and a head group tag for fluorescence microscopy measurements. The accumulation of lipids at curvatures ranging from 28 nm to 55 nm radii was measured and fluorescein labeled DHPE accumulated more than fluorescein labeled HDA at regions of membrane curvature. We then tested whether single biotinylated DHPE molecules sense curvature using single particle tracking methods. Similar to groups of fluorescein labeled DHPE accumulating at curvature, the dynamics of single molecules of biotinylated DHPE was also affected by membrane curvature and highly confined motion was observed.

Fluorescence quantum yield measurements of fluorescent proteins: A laboratory experiment for a biochemistry or molecular biophysics laboratory course

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper‐level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum.

Polymer mediated layer-by-layer assembly of different shaped gold nanoparticles

Gold nanoparticles (GNPs) have a wide range of properties with potential applications in electronics, optics, catalysis, and sensing. In order to demonstrate that dense, stable, and portable samples could be created for these applications, multiple layers of GNPs were assembled via drop casting on glass substrates by layer-by-layer (LBL) techniques. Two cationic polyelectrolytes, poly(diallyldimethylammonium chloride) and polyethyleneimine, one anionic polyelectrolyte, poly(sodium 4-styrene sulfonate), and one neutral polymer, polyvinylpyrrolidone, were combined with four different shapes of GNPs (spherical, rod, triangular prismatic, and octahedral) to prepare thin films. A subset of these polymer nanoparticle combinations were assembled into thin films. Synthesized GNPs were characterized via dynamic light scattering, UV-vis spectroscopy, and transmission electron microscopy and the LBL thin films were characterized using UV-vis spectroscopy and atomic force microscopy. Sensing applications of the nanoparticles in solution and thin films were tested by monitoring the localized surface plasmon resonance of the GNPs. LBL thin films were prepared ranging from 25 to 100 layers with optical densities at plasmon from 0.5 to 3.0. Sensitivity in solutions ranged from 14 to 1002nm/refractive index units (RIU) and films ranged from 18.8 to 135.1nm/RIU suggesting reduced access to the GNPs within the films.

Conformational Changes in C-Reactive Protein Affect Binding to Curved Membranes in a Lipid Bilayer Model of the Apoptotic Cell Surface

C-reactive protein (CRP) is a serum protein that binds to damaged membranes through a phosphatidylcholine binding site. The membrane binding process can initiate the complement immune response and facilitates the clearance of apoptotic cells, likely aiding in the protection of autoimmunity. The initiation of an immune response relies on a conformation change from a native, pentameric form to a modified form, where the modified form binds complement proteins (i.e., C1q) and regulatory proteins substantially better than the native form. In vitro, this reactivity is observed when CRP is monomeric, and a modified form has also been observed at sites of inflammation. Despite evidence that the monomeric form has much higher affinities for almost all proteinaceous binding partners, the role of CRP conformation on lipid binding is yet unknown. In this work, we mimic the outer leaflet of apoptotic cell membranes using a nanopatterned substrate to create curved, supported lipid bilayers and then characterize how CRP conformation affects the interactions between CRP and target membranes. In this assay, the chemical composition and shape are separately tunable parameters. The lipids consisted primarily of palmitoyloleoylphosphatidylcholine, with and without lysophosphatidylcholine, and the curvature had a radius of 27-55 nm. Using this model system combined with quantitative fluorescence microscopy methods, CRP binding to lipid membranes was measured as a function of different conformations of CRP. The modified form of CRP bound curved membranes, but the pentameric form did not for the range of curvatures measured. Unlike most other curvature-sensing proteins, modified CRP accumulated more at a moderate curvature, rather than highly curved or flat regions, suggesting that the membrane bound form does not solely depend on a defect binding mechanism. The presence of lysophosphatidylcholine, a component of apoptotic membranes, increased CRP binding to all types of membranes. Overall, our results show that CRP interactions vary with protein form, lipid composition, and membrane shape. The mechanism by which CRP recognizes damaged membranes depends on the combination of all three.