Coordinated migration and placement of interneurons and projection neurons lead to functional connectivity in the cerebral cortex; defective neuronal migration and the resultant connectivity changes underlie the cognitive defects in a spectrum of neurological disorders. Here we show that primary cilia play a guiding role in the migration and placement of postmitotic interneurons in the developing cerebral cortex, and that this process requires the ciliary protein, Arl13b. Through live imaging of interneuronal cilia we show migrating interneurons display highly dynamic primary cilia and we correlate cilia dynamics with the interneuron’s migratory state. We demonstrate that the guidance cue receptors essential for interneuronal migration localize to interneuronal primary cilia, but their concentration and dynamics are altered in the absence of Arl13b. Expression of Arl13b variants known to cause Joubert syndrome induce defective interneuronal migration, suggesting that defects in cilia-dependent interneuron migration may underlie the neurological defects in Joubert syndrome patients.

Direct monitoring of cell death (i.e., apoptosis and necrosis) during or shortly after treatment is desirable in all cancer therapies to determine the outcome. Further differentiation of apoptosis from necrosis is crucial to optimize apoptosis-favored treatment protocols. We investigated the potential modality of using tissue intrinsic fluorescence chromophore, reduced nicotinamide adenine dinucleotide (NADH), for cell death detection. We imaged the fluorescence lifetime changes of NADH before and after staurosporine (STS)-induced mitochondria-mediated apoptosis and hydrogen peroxide (H2O2)-induced necrosis, respectively, using two-photon fluorescence lifetime imaging in live HeLa cells and 143B osteosarcoma. Time-lapsed lifetime images were acquired at the same site of cells. In untreated cells, the average lifetime of NADH fluorescence was approximately 1.3 ns. The NADH average fluorescence lifetime increased to approximately 3.5 ns within 15 min after 1 microM STS treatment and gradually decreased thereafter. The NADH fluorescence intensity increased within 15 min. In contrast, no significant dynamic lifetime change was found in cells treated with 1 mM H2O2. Our findings suggest that monitoring the NADH fluorescence lifetime may be a valuable noninvasive tool to detect apoptosis and distinguish apoptosis from necrosis for the optimization of apoptosis-favored treatment protocols and other clinical applications.

Abstract. The metabolic changes of human mesenchymal stem cells ͑hMSCs͒ during osteogenic differentiation were accessed by reduced nicotinamide adenine dinucleotide ͑NADH͒ fluorescence lifetime. An increase in mean fluorescence lifetime and decrease in the ratio between free NADH and protein-bound NADH correlated with our previously reported increase in the adenosine triphosphate ͑ATP͒ level of hMSCs during differentiation. These findings suggest that NADH fluorescence lifetime may serve as a new optical biomarker for noninvasive selection of stem cells from differentiated progenies. Keywords: microscopy; fluorescence lifetime; stem cell.Paper 08176L received Jun. 5, 2008; accepted for publication Aug. 14, 2008; published online Oct. 9, 2008. Stem cells give rise to tissue progenitor cells, which can differentiate into specific progenies and have potential use in regenerative medicine, disease treatment, and developmental biology. Efforts have been made to search for reliable biomarkers to identify stem cells ex vivo 1 and in vivo 2 so as to gain a better insight into the biology and physiology of stem cells, as well as to increase the selection efficiency from a given cell pool. However, many of the markers are invasive even in in vivo imaging approaches because stem cells were preloaded ex vivo by radionuclide, ferromagnetic, or reporter labeling, 2 which decreases the clinical usefulness of these methods. Recently, a noninvasive biomarker using proton nuclear magnetic resonance spectroscopy ͑ 1 H-MRS͒ has been identified for detection of neural stem and progenitor cells in the human brain in vivo.3 Although the identity of this 1 H-MRS-detected biomarker is not known, it is suggestive of a metabolic profile of fatty acids. In fact, one generally accepted property of stem cells that differs from their differentiated progenies is a lower metabolic rate accompanied by a lower adenosine triphosphate ͑ATP͒ content. 4 The shift from anaerobic glycolysis to the more efficient mitochondrial oxidative metabolism has been demonstrated in the differentiation of cardiomyocytes 5 and human mesenchymal stem cells ͑hMSCs͒. 6 The preference of stem cells to produce energy by glycolysis instead of oxidative phosphorylation is similar to that of cancer cells, which has been termed the Warburg effect.Optical detection/imaging techniques have been employed to study cell metabolism in a noninvasive manner by monitoring the intrinsic fluorescence signal of reduced nicotinamide adenine dinucleotide ͑NADH͒, a key coenzyme in glycolysis and oxidative metabolism. Two measurement schemes are possible: fluorescence lifetime 7 and fluorescence intensity. 8 In the fluorescence lifetime measurement scheme, a fluorescence decay curve is typically fitted to a twocomponent exponential decay function F͑t͒ = a 1 exp͑−t / 1 ͒ + a 2 exp͑−t / 2 ͒, where 1 and 2 correspond to the short and long fluorescence lifetimes of NADH and were reported to be ϳ400 to 500 ps and ϳ2000 to 2500 ps for free and bound NADH, respectively.7 a 1 and a 2 are the c...

The cyclic calcium release and uptake during calcium oscillation are thought to result from calcium-induced calcium release (CICR); however, it is unclear, especially in nonexcitable cells, how the initial calcium mobilization that triggers CICR occurs. We report here a novel mechanism, other than conventional calcium channels or the phopholipase C-inositol trisphosphate system, for initiating calcium oscillation downstream of integrin signaling. Upon integrin ␣ IIb ␤ 3 binding to fibrinogen ligand or the disintegrin rhodostomin, sodium-proton exchanger NHE1 and sodiumcalcium exchanger NCX1 are actively transported to the plasma membrane, and they become physically coupled to integrin ␣ IIb ␤ 3 . Lipid raft-dependent mechanisms modulate the membrane targeting and formation of the NHE1-integrin ␣ IIb ␤ 3 -NCX1 protein complex. NHE1 and NCX1 within such protein complex are functionally coupled, such that a local increase of sodium concentration caused by NHE1 can drive NCX1 to generate sodium efflux in exchange for calcium influx. The resulting calcium increase inside the cell can then trigger CICR as a prelude to calcium oscillation downstream of integrin ␣ IIb ␤ 3 signaling. Fluorescence resonance energy transfer based on fluorescence lifetime measurements is employed here to monitor the intermolecular interactions among NHE1-integrin ␣ IIb ␤ 3 -NCX1, which could not be properly detected using conventional biochemical assays.In many excitable or nonexcitable cells, the concentration of free intracellular calcium oscillates with a period ranging from a few seconds to a few minutes. Such calcium oscillations are involved in a wide variety of cellular functions (1, 2). It is generally believed that, except for minor variations, the cyclic increase and decrease of calcium results from an autocatalytic release of calcium in a process called calcium-induced calcium release (CICR), 2 followed by a slow negative feedback that terminates calcium release. The cytoplasmic free calcium is then taken up into the organelles to reset the cycle. Despite a general agreement on how calcium oscillation proceeds once the system has been turned on, various different mechanisms have been proposed to explain how the initial calcium mobilization is generated that triggers CICR.As a general rule, calcium entry through voltage-gated channels in electrically excitable cells (3) or through agonist-receptor interactions in nonexcitable cells, such as epithelial cells, hepatocytes, or oocytes (4), is thought to initiate the CICR process (1, 2). Typically, in nonexcitable cells, the binding of an agonist, such as a hormone, a growth factor, or an extracellular matrix, to the corresponding cell surface receptor initiates a series of reactions that end in the activation of phopholipase C (PLC) and the production of the secondary messenger inositol trisphosphate (IP 3 ) (1, 2, 4). IP 3 is thought to induce calcium release from the internal endoplasmic reticulum or mitochondria store, and governs the CICR mechanisms that modulate calcium oscillat...

Microtubule cytoskeletal dynamics of cortical progenitors and astroglial cells play critical roles in the emergence of normal functional organization of cerebral cortex and in disease processes such as tumorigenesis. However, tools to efficiently visualize these events are lacking. Here we describe a mouse genetic model to efficiently visualize and analyze radial progenitors, their astroglial progeny, and the microtubule cytoskeleton of these cells in the developing and adult brain. Using this tool, we demonstrate altered microtubule organization and capture dynamics in adenomatous polyposis coli deficient radial progenitors. Further, using multiphoton microscopy, we show the utility of this tool in real-time imaging of astrocytes in living mouse brain and the short- term stable nature of astrocytes in cerebral cortex. Thus, this model will help explore the dynamics of radial progenitor/astrocyte development or dysfunction and the influence of microtubule functions during these events.

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