Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-02-13
2003-12-30
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06670449
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to compositions and methods for monitoring changes in the local environment inside or outside of cells by detecting optical changes in an environment-sensitive reporter molecule. In particular, this invention relates to compositions which may be specifically incorporated into cells, or may be caused to be produced by them, which produce an optically detectable signal in response to a change in the environment in which the optically sensitive reporter molecule is present. This invention is particularly applicable to detecting the release of substances stored in exocytotic vesicles of cells, such as synaptic vesicles, wherein the compositions of the invention are localized in the vesicles and become exposed to the extracellular space upon release of the contents of the vesicles.
BACKGROUND OF THE INVENTION
Various methods are known for monitoring molecular changes in local environments at a microscopic level. Detection of changes in the microenvironments involving active cells presents a particular challenge because many methods involve destruction or damage to the cells being analyzed. Although significant information has been obtained using electrophysiological techniques, these techniques have limited application for studying complex real time cell physiology and also may damage and thereby alter the cells being recorded. Also, electrophysiology is limited to detecting events involving a change in electrical potential.
Many cell process involve changes in the molecular microenvironment. Such processes include, for example, exocytosis and endocytosis which involves contact between the intracellular and extracellular environments, and changes in ion concentration associated with electrically active cells such as neurons and muscle cells. The release of cellular substances, in particular, is a generalized phenomenon of several cell types which is fundamental to the function of multicellular organisms. Blood cells, endocrine cells and neurons are examples of cell types particularly dependent on such processes. Blood granulocytes, for example, release various mediators of inflammation through exocytosis; endocrine cells release hormones required by other cells; and neurons release neurotransmitters packaged in synaptic vesicles. The ability to detect changes in the microenvironment caused, for example, by the fusion of an exocytotic vesicle with a plasma membrane and contact of the lumenal surface and contents of the vesicle with the extracellular space would provide information useful for understanding and modulating processes which causes release of such cellular substances.
Exocytotic events are particularly important to the proper function of neurons since synaptic transmission is dependent upon the controlled release of synaptic vesicles. Many problems in neurophysiology can be reduced to questions about the location, timing, and magnitude of synaptic activity, including, for instance, the integration of inputs by a single neuron, synaptic plasticity, and pattern classification and storage by neural networks. The study of these and related problems would greatly benefit from a method that allows direct recording from many synapses simultaneously, with the capacity to reliably detect single exocytotic events. Such a method would appear optimal because, on the one hand, central synapses generally transmit information via the fusion of a single synaptic vesicle (1,2), while, on the other hand, the computational power of the nervous system arises from networks containing large numbers of synapses (refs. 3-5).
While current electrophysiological methods allow the activity of individual synapses to be recorded, they do not permit populations to be studied. There is a practical limit to the number of cells that can be impaled simultaneously with intracellular electrodes, and importantly, an invasive method requires an a priori decision on which cells to study, making discovery difficult. Extracellular field recordings with multiple electrodes avoids some of these problems and thereby allows the collective activities of many cells to be measured, but does not permit activity to be ascribed to individual synapses or neurons (6,7). Optical imaging of light emission from fluorescent indicators of membrane potential or intracellular Ca
2+
concentration (7-10) greatly increases spatial resolution but again, does not measure synaptic activity directly. An alternative optical approach that offers a direct gauge of synaptic activity is to load synaptic vesicles with fluorescent dyes and to observe dye release (11,12). However, this method is intrinsically incapable of resolving individual quanta, which can cause only a small decrease in total fluorescence. Despite their limitations, these techniques have opened a window on multicellular phenomena as diverse as the representation of visual scenes by retinal ganglion cells (13) and the emergence of cortical circuits during development (14). Methods that reveal the detailed patterns of synaptic inputs and outputs in entire networks can thus be expected to disclose important new physiological concepts operative at the relatively unexplored interface between cellular and systems neurophysiology.
Due to the importance to physiology of proper exocytotic processes in neurons and other cells types, it is therefore desirable to develop sensitive compositions and methods which can detect changes in the microenvironment inside or outside of cells including quantal exocytotic events in real time. Molecules which can detect changes in microenvironments would be useful as probes of cellular events involving changes in such microenvironments due to movement of molecules in solution or the spacial location of molecules associated with cell membranes. It would be particularly desirable to have available molecules that provided an optical signal upon encountering such a change in the microenvironment.
Various types of molecules have been used in the art for the detection of the presence of other molecular entities. Radiochemical labels have high sensitivity but are hazardous and must be used with appropriate caution. In addition, these labels are not useful for real time localization. Optical labels such as fluorescent molecules or other forms of dyes have also been coupled to molecules to act as reporters for the detection of specific molecular entities. Typically a reporter cable of generating an optical signal is bound to a specific binding molecule which is a member of a ligand binding pair. Such binding molecules are usually antibodies, specific binding proteins, e.g. receptors or peptide hormones which specifically binds a corresponding target ligand. These reporter-ligands are reagents which must be added from the external environment to the system under investigation. Their usefulness is therefore limited by their accessibility to the appropriate molecular target, nonspecific binding or diffusion to inappropriate locations and availability of appropriate binding pairs. Another type of molecular reporter comprises signal generating molecules expressed endogenously by a cell. Several bioluminescent proteins have been reported as useful as detectable labels for optically reporting the presence of a molecular entity.
The green fluorescent protein (GFP) of
Aequora victoria
, for example, is a naturally fluorescent protein with a p-hydroxybenzylideneimidazolone chromophore, created by in vivo cyclization and oxidation of the sequence Ser-Tyr-Gly (positions 65-67). The chromophore's phenolic group, derived from Tyr-66, exists in two states of protonation, which in all likelihood underlie the protein's two main excitation peaks at 395 and 475 nm (ref. 47). Several reports have characterized various bioluminescent proteins. See, for example, Cormier et al., “Recombinant DNA Vectors Capable of Expressing Apoaequorin”, U.S. Pat. No. 5,422,266; Prasher, “Modified Apoaequorin Having Increased Bioluminescent Activity”, U.S. Pat. No. 5,541,309; Cormier et al., “Isolated Renilla Luciferase And Me
De Angelis Dino
Miesenböck Gero
Rothman James E.
Carlson Karen Cochrane
Memorial Sloan-Kettering Cancer Center
Morgan & Finnegan , LLP
Snedden Sheridan
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