An In Vivo Fluorescent Sensor Reveals Intracellular Ins(1,3,4,5)P4Dynamics in Single Cells

Abstract Severe congenital neutropenia (CN) is a rare bone marrow failure syndrome with a high incidence of acute leukemia. In previous studies, we could show that point mutations in the gene for the granulocyte colony-stimulating factor (G-CSF) receptor CSF3R are a highly predictive marker for leukemic development in CN patients. To find out at which stage of hematopoietic development these mutations emerge and how they are propagated during hematopoietic differentiation, we analyzed single cells of different hematopoietic subpopulations from CN patients with CSF3R mutations. We found that CSF3R mutations are not restricted to the myeloid compartment but are also detectable in lymphoid cells, although at a much lower percentage. From our observations, we conclude that CSF3R mutations are acquired in multipotent hematopoietic progenitor cells in CN patients and that they are clonally expanded in myeloid cells expressing the G-CSF receptor due to the growth advantage mediated by the CSF3R mutation.

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Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

Molecular Biology of the Cell ◽

10.1091/mbc.e14-07-1187 ◽

2014 ◽

Vol 25 (22) ◽

pp. 3699-3708 ◽

Cited By ~ 15

Author(s):

Anyimilehidi Mazo-Vargas ◽

Heungwon Park ◽

Mert Aydin ◽

Nicolas E. Buchler

Keyword(s):

Gene Expression ◽

Fluorescent Proteins ◽

Single Cells ◽

Time Lapse ◽

Photon Flux ◽

Luminescence Microscopy ◽

Cell Cycle Genes ◽

Light Excitation ◽

Better Than

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

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Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1989.256.1.g254 ◽

1989 ◽

Vol 256 (1) ◽

pp. G254-G263 ◽

Cited By ~ 20

Author(s):

C. S. Chew ◽

M. Ljungstrom ◽

A. Smolka ◽

M. R. Brown

Keyword(s):

Gastric Mucosa ◽

Primary Culture ◽

Cultured Cells ◽

Single Cells ◽

Parietal Cells ◽

Ultrastructural Changes ◽

Long Term Effects ◽

H2 Receptor Antagonist ◽

Morphological Transformations

A new procedure for isolation and primary culture of gastric parietal cells is described. Parietal cells from rabbit gastric mucosa are enriched to greater than 95% purity by combining a Nycodenz gradient separation with centrifugal elutriation. Cells are plated on the basement membrane matrix, Matrigel, and maintained in culture for at least 1 wk. Parietal cells cultured in this manner remain differentiated, cross-react with monoclonal H+-K+-ATPase antibodies, and respond to histamine, gastrin, and cholinergic stimulation with increased acid production as measured by accumulation of the weak base, [14C]aminopyrine. When stimulated, cultured cells undergo ultrastructural changes in which intracellular canaliculi expand and numerous microvilli are observed. These ultrastructural changes are similar to those previously found to occur in vivo and in acutely isolated parietal cells. Morphological transformations in living cells can also be observed with differential interference contrast optics in the light microscope. After histamine stimulation, intracellular canaliculi gradually expand to form large vacuolar spaces. When the H2 receptor antagonist, cimetidine, is added to histamine-stimulated cells, these vacuoles gradually disappear. The ability to maintain hormonally responsive parietal cells in primary culture should make it possible to study direct, long-term effects of a variety of agonists and antagonists on parietal cell secretory-related activity. These cultured cells should also prove to be useful for the study of calcium transients, ion fluxes, and intracellular pH as related to acid secretion in single cells, particularly since morphological transformations can be used to monitor "physiological" responses at the same time within the same cell.

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Impaired Hair Follicle Morphogenesis and Polarized Keratinocyte Movement upon Conditional Inactivation of Integrin-linked Kinase in the Epidermis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-06-0526 ◽

2008 ◽

Vol 19 (4) ◽

pp. 1462-1473 ◽

Cited By ~ 42

Author(s):

Kerry-Ann Nakrieko ◽

Ian Welch ◽

Holly Dupuis ◽

Dawn Bryce ◽

Agnieszka Pajak ◽

...

Keyword(s):

Hair Follicle ◽

Developmental Stages ◽

Single Cells ◽

Forward Movement ◽

Keratinocyte Proliferation ◽

Cultured Keratinocytes ◽

Conditional Inactivation ◽

Integrin Linked Kinase ◽

Key Aspects

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.

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Cellular and functional defects in a mouse model of heart failure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.6.h3101 ◽

2000 ◽

Vol 279 (6) ◽

pp. H3101-H3112 ◽

Cited By ~ 87

Author(s):

Giovanni Esposito ◽

L. F. Santana ◽

Keith Dilly ◽

Jader Dos Santos Cruz ◽

Lan Mao ◽

...

Keyword(s):

Heart Failure ◽

Adrenergic Receptor ◽

Heart Function ◽

Imaging Techniques ◽

Single Cells ◽

Structural Protein ◽

Muscle Lim Protein ◽

Intracellular Ca ◽

Whole Heart

Heart failure and dilated cardiomyopathy develop in mice that lack the muscle LIM protein (MLP) gene (MLP−/−). The character and extent of the heart failure that occurs in MLP−/− mice were investigated using echocardiography and in vivo pressure-volume (P-V) loop measurements. P-V loop data were obtained with a new method for mice (sonomicrometry) using two pairs of orthogonal piezoelectric crystals implanted in the endocardial wall. Sonomicrometry revealed right-shifted P-V loops in MLP−/−mice, depressed systolic contractility, and additional evidence of heart failure. Cellular changes in MLP−/− mice were examined in isolated single cells using patch-clamp and confocal Ca2+ concentration ([Ca2+]) imaging techniques. This cellular investigation revealed unchanged Ca2+ currents and Ca2+ spark characteristics but decreased intracellular [Ca2+] transients and contractile responses and a defect in excitation-contraction coupling. Normal cellular and whole heart function was restored in MLP−/− mice that express a cardiac-targeted transgene, which blocks the function of β-adrenergic receptor (β-AR) kinase-1 (βARK1). These data suggest that, despite the persistent stimulus to develop heart failure in MLP−/− mice (i.e., loss of the structural protein MLP), downregulation and desensitization of the β-ARs may play a pivotal role in the pathogenesis. Furthermore, this work suggests that the inhibition of βARK1 action may prove an effective therapy for heart failure.

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