Examining Intracellular Organelle Function Using Fluorescent Probes

Abstract

Abstract-Fluorescence microscopy imaging has become one of the most useful techniques to assess the activity of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated. The past 2 decades have seen a tremendous advance in the rational design and development in the nature and selectivity of probes to serve as reporters of the intracellular environment in live cells. These probes range from small organic fluorescent molecules to fluorescent biomolecules and photoproteins ingeniously engineered to follow signaling traffic, sense ionic and nonionic second messengers, and report various kinase activities. These probes, together with recent advances in imaging technology, have enabled significantly enhanced spatial and temporal resolution. This review summarizes some of these developments and their applications to assess intracellular organelle function. Key Words: microscopy Ⅲ calcium Ⅲ redox Ⅲ mitochondria Ⅲ fluorescent proteins R esponses of single cells undergoing physiological activation processes or pathological stresses can be quite heterogeneous in time and space. Measurements of these parameters in bulk (such as in tissue or cellular suspension), using conventional physiological and biochemical methods, can often fail to resolve discrete differences in the kinetics and magnitudes of the responses between cells, as well as complex intracellular and subcellular dynamical processes, critical to understanding how cells actually work. For example, during embryonic development or periods of environmental stress, the decision for a certain cell to undergo apoptosis may be triggered in a moment and distinct from its neighbors, the resulting intracellular processes may proceed along a more or less protracted time frame, and these events may occur heterogeneously among (and inside) other cells. Yet, bulk measures of the progression of apoptosis, such as by DNA-laddering, although undeniably valuable, would show only the overall slow progression with time. All-ornone phenomenon occurring in discrete organelles, such as the mitochondrial permeability transition (MPT), appears as a graded response when examined in bulk suspensions of cells or mitochondria. Similar arguments can be made for resolving questions of Ca 2ϩ signaling and gene expression. Ca and, unless they are synchronized (such as in the beating heart), their occurrence or nature (eg, frequency, amplitude, etc) might not be apparent from ensemble measurements. Significant information about these processes would obviously need to use a single cells studies. Currently, one of the most useful techniques to assess the activities of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated is based on fluorescence microscopy imaging. Each of the various imaging techniques requires that the signaling molecule(s), compartment(s), or organelle(s) be labeled in a specific fashion such that they can be tracked in time...

Strategic localization of heart mitochondrial NOS: a review of the evidence

“…There is a general consensus that iNOS is transiently expressed in the heart during immune responses under stress (8) and, therefore, during pathophysiological conditions of the myocardium such as in ischemia-reperfusion injury (133), septicemia (125), heart failure (146), as well as during aging (138). During the last fifteen years, different groups have used various experimental approaches and reported the presence of mtNOS in the heart (1,10,16,40,45,60,66,72,79,91,115,129,130,140,141,143,147). Regarding the localization of mtNOS, one interesting study provided evidence about the docking of eNOS to the outer mitochondrial membrane in endothelial cells (52).…”

“…2C). Studies of cardiac mtNOS have used various methods including immunohistochemistry (10,66), spectrophotometry (16,30,45,79,115,141,142), radiometry (139,143), fluorometry (40,139,143,147), chemiluminescence (143), and electrochemistry (72). On the other hand, one study reported that the ubiquinone/cyt bc 1 is a site where nitrite is reduced to form NO congeners within mitochondria in a NOS-independent manner (100).…”

“…Opening of the pore causes abrupt mitochondrial depolarization, ATP depletion, and necrotic or apoptotic cell death (12). In this regard, heart and liver mtNOS play important roles in mPTP regulation (40,84,147), suggesting relevance of mtNOS for yet another important mitochondrial function. On the other hand, the existence of alternative NOS-independent mechanisms of NO production that may operate in situations in which conventional NO production is impaired has been described (132).…”

Potential of Mesenchymal Stem Cells for Liver Regeneration

“…We labeled human BM-MSC with CFSE, which irreversible stains the plasma membrane [203,204], or DiD, which labels all cell membranes, membrane-derived vesicles, and intracellular organelles such as mitochondria [205][206][207]. Consequently, pre-immune fetal sheep were IP injected with either CSFE-positive MSC alone or CFSE-positive MSC in combination with DiD-positive MSC.…”

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

“…However, quantitative analysis by this method is labor intensive and is more suitable for experiments using immortalized or primary cell lines. The study of individual mitochondria derived from tissue is significantly more difficult and most methods do not allow for easy identification of mitochondrial subsets concurrently with the evaluation of mitochondrial function 3 .…”