scholarly journals NaNuTrap, a technique for in vivo cell nucleus labelling using nanobodies

Development ◽  
2021 ◽  
Author(s):  
Zsuzsa Ákos ◽  
Leslie Dunipace ◽  
Angelike Stathopoulos
Keyword(s):  
Fluorescent Proteins ◽  
Cell Types ◽  
Cell Counting ◽  
Cell Labelling ◽  
Cell Nuclei ◽  
Maturation Time ◽  
Developmental Processes ◽  
Fluorescent Signal ◽  
Two Photon

In vivo cell labelling is challenging in fast developmental processes because many cell types differentiate more quickly than the maturation time of fluorescent proteins making visualization of these tissues impossible with standard techniques. Here we present a nanobody-based method, Nanobody Nuclear Trap (NaNuTrap), which works with the existing Gal4/UAS system in Drosophila and allows for early in vivo cell nuclei labelling independent of the fluorescent protein's maturation time. This restores the utility of fluorescent proteins that have longer maturation times, such as those used in two-photon imaging, for live imaging of fast or very early developmental processes. We also present a more general application of this system, whereby NaNuTrap can convert cytoplasmic GFP expressed in any existing transgenic fly line into a nuclear label. This nuclear re-localization of the fluorescent signal can improve the utility of the GFP label, for example in cell counting, as well as resulting in a general increase in intensity of the live fluorescent signal. We demonstrate these capabilities of NaNuTrap by effectively tracking subsets of cells during the fast movements associated with gastrulation.

scholarly journals Cellular correlates of gray matter volume changes in magnetic resonance morphometry identified by two-photon microscopy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Livia Asan ◽  
Claudia Falfán-Melgoza ◽  
Carlo A. Beretta ◽  
Markus Sack ◽  
Lei Zheng ◽  
...  
Keyword(s):  
Magnetic Resonance ◽  
Cell Density ◽  
Gray Matter ◽  
Gray Matter Volume ◽  
Cell Nuclei ◽  
Cellular Mechanisms ◽  
Tissue Volume ◽  
Two Photon ◽  
Matter Volume

AbstractMagnetic resonance imaging (MRI) of the brain combined with voxel-based morphometry (VBM) revealed changes in gray matter volume (GMV) in various disorders. However, the cellular basis of GMV changes has remained largely unclear. We correlated changes in GMV with cellular metrics by imaging mice with MRI and two-photon in vivo microscopy at three time points within 12 weeks, taking advantage of age-dependent changes in brain structure. Imaging fluorescent cell nuclei allowed inferences on (i) physical tissue volume as determined from reference spaces outlined by nuclei, (ii) cell density, (iii) the extent of cell clustering, and (iv) the volume of cell nuclei. Our data indicate that physical tissue volume alterations only account for 13.0% of the variance in GMV change. However, when including comprehensive measurements of nucleus volume and cell density, 35.6% of the GMV variance could be explained, highlighting the influence of distinct cellular mechanisms on VBM results.

scholarly journals Targeted two-photon chemical apoptotic ablation of defined cell types in vivo

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Robert A. Hill ◽  
Eyiyemisi C. Damisah ◽  
Fuyi Chen ◽  
Alex C. Kwan ◽  
Jaime Grutzendler
Keyword(s):  
Cell Types ◽  
Two Photon

scholarly journals Human CD62Ldim neutrophils identified as a separate subset by proteome profiling and in vivo pulse-chase labeling

Blood ◽  
2017 ◽  
Vol 129 (26) ◽  
pp. 3476-3485 ◽  
Author(s):  
Tamar Tak ◽  
Patrick Wijten ◽  
Marjolein Heeres ◽  
Peter Pickkers ◽  
Arjen Scholten ◽  
...  
Keyword(s):  
Acute Inflammation ◽  
Low Dose ◽  
Cell Types ◽  
Direct Result ◽  
Maturation Time ◽  
Segmented Nucleus ◽  
Proteome Level ◽  
Blood Neutrophils ◽  
Lps Activation

Abstract During acute inflammation, 3 neutrophil subsets are found in the blood: neutrophils with a conventional segmented nucleus, neutrophils with a banded nucleus, and T-cell–suppressing CD62Ldim neutrophils with a high number of nuclear lobes. In this study, we compared the in vivo kinetics and proteomes of banded, mature, and hypersegmented neutrophils to determine whether these cell types represent truly different neutrophil subsets or reflect changes induced by lipopolysaccharide (LPS) activation. Using in vivo pulse-chase labeling of neutrophil DNA with 6,6-2H2-glucose, we found that 2H-labeled banded neutrophils appeared much earlier in blood than labeled CD62Ldim and segmented neutrophils, which shared similar label kinetics. Comparison of the proteomes by cluster analysis revealed that CD62Ldim neutrophils were clearly separate from conventional segmented neutrophils despite having similar kinetics in peripheral blood. Interestingly, the conventional segmented cells were more related at a proteome level to banded cells despite a 2-day difference in maturation time. The differences between CD62Ldim and mature neutrophils are unlikely to have been a direct result of LPS-induced activation, because of the extremely low transcriptional capacity of CD62Ldim neutrophils and the fact that neutrophils do not directly respond to the low dose of LPS used in the study (2 ng/kg body weight). Therefore, we propose CD62Ldim neutrophils are a truly separate neutrophil subset that is recruited to the bloodstream in response to acute inflammation. This trial was registered at www.clinicaltrials.gov as #NCT01766414.

scholarly journals Development of an Immunologically Tolerated Combination of Fluorescent Proteins for In vivo Two-photon Imaging

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Selamawit Gossa ◽  
Debasis Nayak ◽  
Bernd H. Zinselmeyer ◽  
Dorian B. McGavern
Keyword(s):  
Fluorescent Proteins ◽  
Two Photon ◽  
Photon Imaging ◽  
Two Photon Imaging

In vivo expression patterns of MyoD, p21, and Rb proteins in myonuclei and satellite cells of denervated rat skeletal muscle

2004 ◽  
Vol 287 (2) ◽  
pp. C484-C493 ◽  
Author(s):  
Minenori Ishido ◽  
Katsuya Kami ◽  
Mitsuhiko Masuhara
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cell ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Expression Patterns ◽  
Cell Types ◽  
Cell Nuclei ◽  
Myogenic Regulatory Factor

MyoD, a myogenic regulatory factor, is rapidly expressed in adult skeletal muscles in response to denervation. However, the function(s) of MyoD expressed in denervated muscle has not been adequately elucidated. In vitro, it directly transactivates cyclin-dependent kinase inhibitor p21 (p21) and retinoblastoma protein (Rb), a downstream target of p21. These factors then act to regulate cell cycle withdrawal and antiapoptotic cell death. Using immunohistochemical approaches, we characterized cell types expressing MyoD, p21, and Rb and the relationship among these factors in the myonucleus of denervated muscles. In addition, we quantitatively examined the time course changes and expression patterns among distinct myofiber types of MyoD, p21, and Rb during denervation. Denervation induced MyoD expression in myonuclei and satellite cell nuclei, whereas p21 and Rb were found only in myonuclei. Furthermore, coexpression of MyoD, p21, and Rb was induced in the myonucleus, and quantitative analysis of these factors determined that there was no difference among the three myofiber types. These observations suggest that MyoD may function in myonuclei in response to denervation to protect against denervation-induced apoptosis via perhaps the activation of p21 and Rb, and function of MyoD expressed in satellite cell nuclei may be negatively regulated. The present study provides a molecular basis to further understand the function of MyoD expressed in the myonuclei and satellite cell nuclei of denervated skeletal muscle.

scholarly journals Distinct features of brain perivascular fibroblasts and mural cells revealed by in vivo two-photon imaging

2021 ◽  
pp. 0271678X2110685
Author(s):  
Stephanie K Bonney ◽  
Liam T Sullivan ◽  
Timothy J Cherry ◽  
Richard Daneman ◽  
Andy Y Shih
Keyword(s):  
Cell Types ◽  
Structural Features ◽  
Main Trunk ◽  
Perivascular Niche ◽  
Two Photon ◽  
Mural Cells ◽  
Adult Mice ◽  
Photon Imaging ◽  
Two Photon Imaging

Perivascular fibroblasts (PVFs) are recognized for their pro-fibrotic role in many central nervous system disorders. Like mural cells, PVFs surround blood vessels and express Pdgfrβ. However, these shared attributes hinder the ability to distinguish PVFs from mural cells. We used in vivo two-photon imaging and transgenic mice with PVF-targeting promoters (Col1a1 or Col1a2) to compare the structure and distribution of PVFs and mural cells in cerebral cortex of healthy, adult mice. We show that PVFs localize to all cortical penetrating arterioles and their offshoots (arteriole-capillary transition zone), as well as the main trunk of only larger ascending venules. However, the capillary zone is devoid of PVF coverage. PVFs display short-range mobility along the vessel wall and exhibit distinct structural features (flattened somata and thin ruffled processes) not seen with smooth muscle cells or pericytes. These findings clarify that PVFs and mural cells are distinct cell types coexisting in a similar perivascular niche.

scholarly journals Single-Cell Techniques Using Chromosomally Tagged Fluorescent Bacteria To Study Listeria monocytogenes Infection Processes

2010 ◽  
Vol 76 (11) ◽  
pp. 3625-3636 ◽  
Author(s):  
Damien Balestrino ◽  
M�lanie Anne Hamon ◽  
Laurent Dortet ◽  
Marie-Anne Nahori ◽  
Javier Pizarro-Cerda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Listeria Monocytogenes ◽  
Single Cell ◽  
Fluorescent Proteins ◽  
Cell Types ◽  
Intracellular Replication ◽  
Infectious Process ◽  
Infection Processes

ABSTRACT Listeria monocytogenes is a Gram-positive facultative intracellular pathogen which invades different cell types, including nonphagocytic cells, where it is able to replicate and survive. The different steps of the cellular infectious process have been well described and consist of bacterial entry, lysis of the endocytic vacuole, intracellular replication, and spreading to neighboring cells. To study the listerial infectious process, gentamicin survival assays, plaque formation, and direct microscopy observations are typically used; however, there are some caveats with each of these techniques. In this study we describe new single-cell techniques based on use of an array of integrative fluorescent plasmids (green, cyan, and yellow fluorescent proteins) to easily, rapidly, and quantitatively detect L. monocytogenes in vitro and in vivo. We describe construction of 13 integrative and multicopy plasmids which can be used for detecting intracellular bacteria, for measuring invasion, cell-to-cell spreading, and intracellular replication, for monitoring in vivo infections, and for generating transcriptional or translational reporters. Furthermore, we tested these plasmids in a variety of epifluorescence- and flow cytometry-based assays. We showed that we could (i) determine the expression of a particular promoter during the cell cycle, (ii) establish in one rapid experiment at which step in the cell cycle a particular mutant is defective, and (iii) easily measure the number of infected cells in vitro and in mouse organs. The plasmids that are described and the methods to detect them are new powerful tools to study host-Listeria interactions in a fast, robust, and high-throughput manner.

scholarly journals Chronic imaging of mitochondria in the murine cerebral vasculature using in vivo two-photon microscopy

2020 ◽  
Vol 318 (6) ◽  
pp. H1379-H1386
Author(s):  
Ibolya Rutkai ◽  
Wesley R. Evans ◽  
Nikita Bess ◽  
Tomas Salter-Cid ◽  
Siniša Čikić ◽  
...  
Keyword(s):  
Cerebral Circulation ◽  
Three Dimensional ◽  
Cell Types ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Dimensional Reconstruction ◽  
Different Cell Types

We introduce an innovative in vivo approach to study mitochondria in the cerebral circulation in their physiological environment by demonstrating the feasibility of long-term imaging and three-dimensional reconstruction. We postulate that the appropriate combination of Cre/Lox system and two-photon microscopy will contribute to a better understanding of the role of mitochondria in not only endothelium but also the different cell types of the cerebral circulation.

scholarly journals Temporal dynamics of inhalation-linked activity across defined subpopulations of mouse olfactory bulb neurons imaged in vivo

2019 ◽  
Author(s):  
Shaina M. Short ◽  
Matt Wachowiak
Keyword(s):  
Olfactory Bulb ◽  
Temporal Dynamics ◽  
Cell Types ◽  
Response Patterns ◽  
Two Photon ◽  
Cell Type Specific ◽  
Olfactory Processing ◽  
Tufted Cells ◽  
Cell Subpopulations

ABSTRACTIn mammalian olfaction, inhalation drives the temporal patterning of neural activity that underlies early olfactory processing, and a single inhalation of odorant is sufficient for odor perception. However, how the neural circuits that process incoming olfactory information are activated in the context of inhalation-linked dynamics remains poorly understood. To better understand early olfactory processing in vivo, we used an artificial inhalation paradigm combined with two-photon calcium imaging to compare the dynamics of activity evoked by odorant inhalation across major cell types of the mouse olfactory bulb. Transgenic models and cell-type specific genetic tools were used to express GCaMP6f or jRGECO1a in mitral and tufted cell subpopulations, olfactory sensory neurons and two major juxtaglomerular interneuron classes, and responses to a single inhalation of odorant were compared. Activity in all cell types was strongly linked to inhalation, and all cell types showed some variance in the latency, rise-times and durations of their inhalation-linked response patterns. The dynamics of juxtaglomerular interneuron activity closely matched that of sensory neuron inputs, while mitral and tufted cells showed the highest diversity in dynamics, with a range of latencies and durations that could not be accounted for by heterogeneity in the dynamics of sensory input. Surprisingly, temporal response patterns of mitral and superficial tufted cells were highly overlapping such that these two subpopulations could not be distinguished on the basis of their inhalation-linked dynamics, with the exception of a subpopulation of superficial tufted cells expressing the peptide transmitter cholecystokinin. Overall, these results support a model in which diversity in inhalation-linked patterning of OB output arises first at the level of OSN inputs to the OB and is enhanced by feedforward inhibition from juxtaglomerular interneurons which differentially impacts different subpopulations of OB output neurons.

scholarly journals Distinct features of brain perivascular fibroblasts and mural cells revealed by in vivo two-photon imaging

2021 ◽  
Author(s):  
Stephanie K Bonney ◽  
Liam T Sullivan ◽  
Timothy Joel Cherry ◽  
Richard Daneman ◽  
Andy Y Shih
Keyword(s):  
Cell Types ◽  
Structural Features ◽  
Main Trunk ◽  
Perivascular Niche ◽  
Two Photon ◽  
Mural Cells ◽  
Adult Mice ◽  
Photon Imaging ◽  
Two Photon Imaging

Perivascular fibroblasts (PVFs) are recognized for their pro-fibrotic role in many central nervous system disorders. Like mural cells, PVFs surround blood vessels and express Pdgfrβ. However, these shared attributes hinder the ability to distinguish PVFs from mural cells. We used in vivo two-photon imaging and transgenic mice with PVF-targeting promoters (Col1a1 or Col1a2) to compare the structure and distribution of PVFs and mural cells in cerebral cortex of healthy, adult mice. We show that PVFs localize to all cortical penetrating arterioles and their pre-capillary offshoots, as well as the main trunk of only larger ascending venules. However, the capillary zone is devoid of PVF coverage. PVFs display short-range mobility along the vessel wall and exhibit distinct structural features (flattened somata and thin ruffled processes) not seen with smooth muscle cells or pericytes. These findings clarify that PVFs and mural cells are distinct cell types coexisting in a similar perivascular niche.

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