Coordination of force-generating actin-based modules stabilizes and remodels membranes in vivo.

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step.

Spatial and Temporal Coordination of Force-generating Actin-based Modules Drives Membrane Remodeling .

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that: a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane; and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step.

Loss of Cdc42 in Exocrine Acini Decreases Saliva Secretion but Increases Tear Secretion-A Potential Model of Exocrine Gland Senescence.

Cdc42 is a small GTPase essential for the cell cycle, morphogenesis, and cell adhesion, and it is involved in the polarity of epithelial cells. However, the functional roles of Cdc42 in exocrine glands, such as the maintenance of acini and water secretion, are not yet well understood. In this study, we generated acinar-cell-specific conditional knockout () mice to assess their maintenance of acinar cells and physiological functions in the salivary glands (SGs) and lacrimal glands (LGs).

Insertion of circularly permuted cyan fluorescent protein into the ligand-binding domain of inositol 1,4,5-trisphosphate receptor for enhanced FRET upon binding of fluorescent ligand.

Binding of fluorescent ligand (FL) to the cyan fluorescent protein (CFP)-coupled ligand-binding domain of the inositol 1,4,5-trisphosphate (IP) receptor (CFP-LBP) produces fluorescence (Förster) resonance energy transfer (FRET). A competitive fluorescent ligand assay (CFLA), using the FRET signal from competition between FLs and IP, can measure IP concentration. The FRET signal should be enhanced by attaching a FRET donor to an appropriate position.

Retinal damage alters gene expression profile in lacrimal glands of mice.

Early detection of such retinal diseases as glaucoma and age-related macular degeneration (AMD) is important to prevent blindness. There have been reports of changes in some components in the tears of glaucoma and AMD patients, suggesting tears' potential usefulness in screening for retinal diseases. We hypothesized that retinal damage might alter gene expression in the lacrimal gland, leading to those changes in tear components.

Transduction of Salivary Gland Acinar Cells with a Novel AAV Vector 44.9.

The loss of salivary gland function caused by radiation therapy of the head and neck or autoimmune disease such as Sjögren's syndrome is a serious condition that affects a patient's quality of life. Due to the combined exocrine and endocrine functions of the salivary gland, gene transfer to the salivary glands holds the potential for developing therapies for disorders of the salivary gland and the expression of therapeutic proteins via the exocrine pathway to the mouth, upper gastrointestinal tract, or endocrine pathway, systemically, into the blood. Recent clinical success with viral vector-mediated gene transfer for the treatment of irradiation-induced damage to the salivary glands has highlighted the need for the development of novel vectors with acinar cell tropism able to result in stable long-term transduction.

Cryopreservation of Biologically Functional Submandibular Gland Rudiments from Fetal Mice.

Background/aim: Cryopreservation of cell lines has been widely used in the laboratory; however, cryopreservation of organs is still considered to be difficult. The submandibular gland (SMG) of fetal mice is one of the best-characterized organs. We investigated the conditions for cryopreserving SMG rudiments.

Arginase 1 is involved in lacrimal hyposecretion in male NOD mice, a model of Sjögren's syndrome, regardless of dacryoadenitis status.

Key Points: Few reports have explored the possibility of involvement of non-inflammatory factors in lacrimal hyposecretion in Sjögren's syndrome (SS). RNA-sequencing analysis revealed that only four genes, including arginase 1, were downregulated in the lacrimal gland of SS model male mice (NOD mice) after onset of lacrimal hyposecretion and dacryoadenitis. Even in non-dacryoadenitis-type NOD mice, tear secretion and arginase 1 expression remained low.

Cdc42 controls secretory granules morphology in rodent salivary glands in vivo.

We previously reported that the small GTPase Cdc42 negatively regulates endocytosis in the salivary gland of live mice. By using intravital subcellular microscopy, we showed that depletion of Cdc42 causes the mis-sorting of plasma membrane components into intracellular vesicles, ultimately leading to the impairment of the homeostasis of the apical plasma membrane. In this study, we report that, besides, Cdc42 depletion alters the ultrastructure of large secretory granules analyzed by transmission electron microscopy.

Cdc42 negatively regulates endocytosis during apical membrane maintenance in live animals.

Lumen establishment and maintenance are fundamental for tubular organs physiological functions. Most of the studies investigating the mechanisms regulating this process have been carried out in cell cultures or in smaller organisms, whereas little has been done in mammalian model systems in vivo. Here we used the salivary glands of live mice to examine the role of the small GTPase Cdc42 in the regulation of the homeostasis of the intercellular canaliculi, a specialized apical domain of the acinar cells, where protein and fluid secretion occur.

Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals.

Membrane remodeling plays a fundamental role during a variety of biological events. However, the dynamics and the molecular mechanisms regulating this process within cells in mammalian tissues in situ remain largely unknown. In this study, we use intravital subcellular microscopy in live mice to study the role of the actomyosin cytoskeleton in driving the remodeling of membranes of large secretory granules, which are integrated into the plasma membrane during regulated exocytosis.

Imaging membrane remodeling during regulated exocytosis in live mice.

In this mini-review we focus on the use of time-lapse light microscopy to study membrane remodeling during protein secretion in live animals. In particular, we highlight how subcellular intravital microscopy has enabled imaging the dynamics of both individual secretory vesicles and the plasma membrane, during different steps in the exocytic process. This powerful approach has provided us with the unique opportunity to unravel the role of the actin cytoskeleton in regulating this process under physiological conditions, and to overcome the shortcomings of more reductionist model systems.

Polyethylenimine-mediated expression of transgenes in the acinar cells of rats salivary glands in vivo.

Non viral-mediated transfection of plasmid DNA provides a fast and reliable way to express various transgenes in selected cell populations in live animals. Here, we show an improvement of a previously published method that is based on injecting plasmid DNA into the ductal system of the salivary glands in live rats. Specifically, using complexes between plasmid DNA and polyethyleneimine (PEI) we show that the expression of the transgenes is directed selectively to the salivary acinar cells.

In vivo tissue-wide synchronization of mitochondrial metabolic oscillations.

Little is known about the spatiotemporal coordination of mitochondrial metabolism in multicellular organisms in situ. Using intravital microscopy in live animals, we report that mitochondrial metabolism undergoes rapid and periodic oscillations under basal conditions. Notably, mitochondria in vivo behave as a network of functionally coupled oscillators, which maintain a high level of coordination throughout the tissue via the activity of gap junctions.

Probing the role of the actin cytoskeleton during regulated exocytosis by intravital microscopy.

The actin cytoskeleton plays a fundamental role in controlling several steps during regulated exocytosis. Here, we describe a combination of procedures that are aimed at studying the dynamics and the mechanism of the actin cytoskeleton in the salivary glands of live rodents, a model for exocrine secretion. Our approach relies on intravital microscopy, an imaging technique that enables imaging biological events in live animals at a subcellular resolution, and it is complemented by the use of pharmacological agents and indirect immunofluorescence in the salivary tissue.

VAMP4 is required to maintain the ribbon structure of the Golgi apparatus.

The Golgi apparatus forms a twisted ribbon-like network in the juxtanuclear region of vertebrate cells. Vesicle-associated membrane protein 4 (VAMP4), a v-SNARE protein expressed exclusively in the vertebrate trans-Golgi network (TGN), plays a role in retrograde trafficking from the early endosome to the TGN, although its precise function within the Golgi apparatus remains unclear. To determine whether VAMP4 plays a functional role in maintaining the structure of the Golgi apparatus, we depleted VAMP4 gene expression using RNA interference technology.

Isoproterenol stimulates transient SNAP23-VAMP2 interaction in rat parotid glands.

The exocytosis of salivary proteins is mainly regulated by cAMP, although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which mediate cAMP-dependent exocytic membrane fusion, have remained unidentified. Here we examined the effect of isoproterenol (ISO) and cytochalasin D (CyD) on the level of SNARE complexes in rat parotid glands. When SNARE complexes were immunoprecipitated by anti-SNAP23, the coprecipitation of VAMP2 was significantly increased in response to ISO and/or CyD, although the coprecipitation of VAMP8 or syntaxin 4 was scarcely augmented.

SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells.

To evaluate the role of SNARE proteins in the constitutive exocytosis, we knocked down syntaxin 3, 4, 5, 6, 7, and VAMP3, 5, 7, 8 with their siRNAs, and determined the cell-to-medium ratio of CLuc, a secreted luciferase of Cypridina noctiluca. Although the protein level of SNAREs in HeLa cells was markedly reduced by the siRNA treatment, the cell/medium ratio was scarcely increased by any siRNAs except for syntaxin 5. The accumulation of GFP-tagged human growth hormone was also visible only by the knockdown of syntaxin 5.

Expression of functional Stim1-mKO1 in rat submandibular acinar cells by retrograde ductal injection of an adenoviral vector.

Objective: Adenoviruses are used for gene transfer to salivary glands cells in vivo. We constructed an adenovirus vector that expressed a fusion protein of human Stim1 and the fluorescent protein mKO1 (Ad-Stim1-mKO1), and used it to investigate the molecular dynamics and functions of exogenously expressed proteins in living salivary acinar cells.

Design: Ad-Stim1-mKO1 was transferred to rat submandibular glands by retrograde ductal injection.

Spontaneous oscillations in intracellular Ca(2+) concentration via purinergic receptors elicit transient cell swelling in rat parotid ducts.

Using multiphoton microscopy, we established that rat parotid ductal cells exhibit spontaneous oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). These oscillatory Ca(2+) responses were observed during continuous perfusion with a physiological salt solution at 37 degrees C in the absence of calcium mobilizing agonist stimulation. The timing and patterns of these spontaneous Ca(2+) oscillations varied among individual ductal cells, and the average number of Ca(2+) responses in a single responding ductal cell was 2.

Use of Fluorescence Resonance Energy Transfer-based Biosensors for the Quantitative Analysis of Inositol 1,4,5-Trisphosphate Dynamics in Calcium Oscillations.

Inositol 1,4,5-trisphosphate (IP(3)) is an intracellular messenger that elicits a wide range of spatial and temporal Ca(2+) signals, and this signaling versatility is exploited to regulate diverse cellular responses. In this study, we have developed a series of IP(3) biosensors that exhibit strong pH stability and varying affinities for IP(3), as well as a method for the quantitative measurement of cytosolic concentrations of IP(3) ([IP(3)](i)) in single living cells. We applied this method to elucidate IP(3) dynamics during agonist-induced Ca(2+) oscillations, and we demonstrated cell type-dependent differences in IP(3) dynamics, a nonfluctuating rise in [IP(3)](i) and repetitive IP(3) spikes during Ca(2+) oscillations in COS-7 cells and HSY-EA1 cells, respectively.

Spontaneous Ca2+ oscillations via purinergic receptors elicit transient cell swelling in rat parotid ducts.

Rat parotid ductal cells were found to exhibit spontaneous Ca(2+) oscillations. These oscillatory Ca(2+) responses were observed during continuous perfusion with physiological salt solution at 37 degrees C in the absence of calcium mobilizing agonist stimulation. These Ca(2+) oscillations were completely blocked by the purinergic receptor inhibitors, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) (PPADS) and suramin, but were not blocked by the muscarinic antagonist, atropine, nor the alpha-adrenergic antagonist, phentolamine.

Multi-photon microscopic imaging of rat parotid ducts demonstrates cellular heterogeneity in Ca2+ responsiveness.

The heterogeneity of salivary ductal cells, with regard to their sensitivity to Ca(2+)-mobilizing agonists, was visualized by multi-photon microscopy. Stimulation of isolated parotid ducts with 0.1 and 1 microM epinephrine (Epi) elevated the intracellular Ca(2+) levels ([Ca(2+)](i)) in approximately 30% and >90% of the ductal cells, respectively.

A novel fluorescent method employing the FRET-based biosensor "LIBRA" for the identification of ligands of the inositol 1,4,5-trisphosphate receptors.

LIBRA is a fluorescent biosensor of inositol 1,4,5-trisphosphate (IP(3)) and is composed of the ligand-binding domain of the rat type 3 IP(3) receptor and cyan and yellow fluorescent proteins. We examined the responses of LIBRA and its IP(3)-insensitive mutant LIBRA-N to compounds known to inhibit IP(3)-induced Ca(2+) release. Heparin, a competitive antagonist of IP(3) receptors, increased the emission ratio of LIBRA but not that of LIBRA-N.