Measuring Metabolic Changes in Cancer Cells Using Two-Photon Fluorescence Lifetime Imaging Microscopy and Machine-Learning Analysis.

Two-photon (2P) fluorescence lifetime imaging microscopy (FLIM) was used to track cellular metabolism with drug treatment of auto-fluorescent coenzymes NAD(P)H and FAD in living cancer cells. Simultaneous excitation at 800 nm of both coenzymes was compared with traditional sequential 740/890 nm plus another alternative of 740/800 nm, before and after adding doxorubicin in an imaging time course. Changes of redox states at single cell resolution were compared by three analysis methods: our recently introduced fluorescence lifetime redox ratio (FLIRR: NAD(P)H-a %/FAD-a %), machine-learning (ML) algorithms using principal component (PCA) and non-linear multi-Feature autoencoder (AE) analysis.

Chromokinesin Klp-19 regulates microtubule overlap and dynamics during anaphase in .

Recent studies have highlighted the significance of the spindle midzone, the region between the segregating chromosomes, in ensuring proper chromosome segregation. By combining 3D electron tomography, cutting-edge light microscopy and a novel single cell essay allowing single molecule tracking, we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of by the chromokinesin KLP-19, and its relevance for proper spindle function. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1.

Extracellular Tau Oligomers Damage the Axon Initial Segment.

Background: In Alzheimer's disease (AD) brain, neuronal polarity and synaptic connectivity are compromised. A key structure for regulating polarity and functions of neurons is the axon initial segment (AIS), which segregates somatodendritic from axonal proteins and initiates action potentials. Toxic tau species, including extracellular oligomers (xcTauOs), spread tau pathology from neuron to neuron by a prion-like process, but few other cell biological effects of xcTauOs have been described.

Characterization of mitochondrial dysfunction due to laser damage by 2-photon FLIM microscopy.

Mitochondria are the central organelles in cellular bio-energetics with key roles to play in energy metabolism and cell fate decisions. Fluorescence Lifetime Imaging microscopy (FLIM) is used to track metabolic changes by following the intrinsic co-enzymes NAD(P)H and FAD, present in metabolic pathways. FLIM records-lifetimes and the relative fractions of free (unbound) and bound states of NAD(P)H and FAD are achieved by multiphoton excitation of a pulsed femto-second infra-red laser.

Optimization of FLIM imaging, fitting and analysis for auto-fluorescent NAD(P)H and FAD in cells and tissues.

Increasingly, the auto-fluorescent coenzymes NAD(P)H and FAD are being tracked by multi-photon fluorescence lifetime microscopy (FLIM) and used as versatile markers for changes in mammalian metabolism. The cellular redox state of different cell model systems, organoids and tissue sections is investigated in a range of pathologies where the metabolism is disrupted or reprogrammed; the latter is particularly relevant in cancer biology. Yet, the actual optimized process of acquiring images by FLIM, execute a correct lifetime fitting procedure and subsequent processing and analysis can be challenging for new users.

Intraneuronal Tau Misfolding Induced by Extracellular Amyloid-β Oligomers.

Abnormal folding and aggregation of the microtubule-associated protein, tau, is a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD). Although normal tau is an intrinsically disordered protein, it does exhibit tertiary structure whereby the N- and C-termini are often in close proximity to each other and to the contiguous microtubule-binding repeat domains that extend C-terminally from the middle of the protein. Unfolding of this paperclip-like conformation might precede formation of toxic tau oligomers and filaments, like those found in AD brain.

Single-cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H.

Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state.

The Ancient Genetic Networks of Obesity: Whole-Animal Automated Screening for Conserved Fat Regulators.

Caenorhabditis elegans is the first and only metazoan model that enables whole-body gene knockdown by simply feeding their standard laboratory diet, E. coli, carrying RNA interference (RNAi)-expressing constructs. The simplicity of the RNAi treatment, small size, and fast reproduction rate of C.

Segmented cell analyses to measure redox states of autofluorescent NAD(P)H, FAD & Trp in cancer cells by FLIM.

Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio.

An Hdac1/Rpd3-Poised Circuit Balances Continual Self-Renewal and Rapid Restriction of Developmental Potential during Asymmetric Stem Cell Division.

How the developmental potential of differentiating stem cell progeny becomes rapidly and stably restricted following asymmetric stem cell division is unclear. In the fly larval brain, earmuff (erm) uniquely functions to restrict the developmental potential of intermediate neural progenitors (INPs) generated by asymmetrically dividing neural stem cells (neuroblasts). Here we demonstrate that the histone deacetylase Hdac1/Rpd3 functions together with self-renewal transcriptional repressors to maintain the erm immature INP enhancer in an inactive but poised state in neuroblasts.

Neuroblast niche position is controlled by Phosphoinositide 3-kinase-dependent DE-Cadherin adhesion.

Correct positioning of stem cells within their niche is essential for tissue morphogenesis and homeostasis. How stem cells acquire and maintain niche position remains largely unknown. Here, we show that a subset of brain neuroblasts (NBs) in utilize Phosphoinositide 3-kinase (PI3-kinase) and DE-cadherin to build adhesive contact for NB niche positioning.

Spindle orientation during asymmetric cell division.

Development of a multicellular organism from a fertilized egg depends on a precise balance between symmetric cell divisions to expand the pool of similar cells, and asymmetric cell divisions to create cell-type diversity. Spindle orientation can influence the generation of symmetric or asymmetric cell fates depending on how it is coupled to cell-intrinsic polarity cues, or how it is positioned relative to cell-extrinsic cues such as niche-derived signals. In this review, we describe the mechanism of spindle orientation in budding yeast, Drosophila melanogaster, Caenorhabditis elegans and mammalian neural progenitors, with the goal of highlighting conserved mechanisms and indicating open questions for the future.

Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts.

Mitotic spindle orientation in polarized cells determines whether they divide symmetrically or asymmetrically. Moreover, regulated spindle orientation may be important for embryonic development, stem cell biology, and tumor growth. Drosophila neuroblasts align their spindle along an apical/basal cortical polarity axis to self-renew an apical neuroblast and generate a basal differentiating cell.

Galphai generates multiple Pins activation states to link cortical polarity and spindle orientation in Drosophila neuroblasts.

Drosophila neuroblasts divide asymmetrically by aligning their mitotic spindle with cortical cell polarity to generate distinct sibling cell types. Neuroblasts asymmetrically localize Galphai, Pins, and Mud proteins; Pins/Galphai direct cortical polarity, whereas Mud is required for spindle orientation. It is currently unknown how Galphai-Pins-Mud binding is regulated to link cortical polarity with spindle orientation.

The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts.

Asymmetric cell division generates cell diversity during development and regulates stem-cell self-renewal in Drosophila and mammals. In Drosophila, neuroblasts align their spindle with a cortical Partner of Inscuteable (Pins)-G alpha i crescent to divide asymmetrically, but the link between cortical polarity and the mitotic spindle is poorly understood. Here, we show that Pins directly binds, and coimmunoprecipitates with, the NuMA-related Mushroom body defect (Mud) protein.

Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function.

Abstrakt, a DEAD box protein, regulates Insc levels and asymmetric division of neural and mesodermal progenitors.

Asymmetric cell division generates cell diversity in bacteria, yeast, and higher eukaryotes. In Drosophila, both neural and muscle progenitors divide asymmetrically. In these cells the Inscuteable (Insc) protein complex coordinates cell polarity and spindle orientation.