scholarly journals Actin dynamics drive microvillar motility and clustering during brush border assembly

2018 ◽  
Author(s):  
Leslie M Meenderink ◽  
Matthew J. Tyska
Keyword(s):  
Epithelial Cells ◽  
Brush Border ◽  
Driving Force ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Actin Dynamics ◽  
Apical Surface ◽  
Actin Assembly ◽  
Brush Borders ◽  
Surface Remodeling

SUMMARYDuring differentiation, transporting epithelial cells generate large arrays of microvilli known as a brush borders to enhance functional capacity. To develop our understanding of brush border formation, we used live cell imaging to visualize apical surface remodeling during early stages of this process. Strikingly, we found that individual microvilli exhibit persistent active motility, translocating across the cell surface at ~0.2 μm/min. Perturbation studies with inhibitors and photokinetic experiments revealed that microvillar motility is driven by actin assembly at the barbed-ends of core bundles, which in turn is linked to robust treadmilling of these structures. Because the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately leads to their clustering and consolidation into higher order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.

scholarly journals Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging

2006 ◽  
Vol 172 (7) ◽  
pp. 1035-1044 ◽  
Author(s):  
Wei Hua ◽  
David Sheff ◽  
Derek Toomre ◽  
Ira Mellman
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Basolateral Membrane ◽  
Live Cell ◽  
Junctional Complex ◽  
Apical Surface

Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin–Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi network (TGN) exit, passage through transport intermediates, and arrival at the plasma membrane using cyan/yellow fluorescent protein–tagged glycosylphosphatidylinositol-anchored protein and vesicular stomatitis virus glycoprotein as apical and basolateral reporters, respectively. For both pathways, exit from the TGN was rate limiting. Furthermore, apical and basolateral proteins were targeted directly to their respective membranes, resolving current confusion as to whether sorting occurs on the secretory pathway or only after endocytosis. However, a transcytotic protein did reach the apical surface after a prior appearance basolaterally. Finally, newly synthesized proteins appeared to be delivered to the entire lateral or apical surface, suggesting—contrary to expectations—that there is not a restricted site for vesicle docking or fusion adjacent to the junctional complex.

Live-Cell Imaging of F-Actin Dynamics During Fertilization in Arabidopsis thaliana

2017 ◽  
pp. 47-54 ◽  
Author(s):  
Daichi Susaki ◽  
Daisuke Maruyama ◽  
Ramesh Yelagandula ◽  
Frederic Berger ◽  
Tomokazu Kawashima
Keyword(s):  
Arabidopsis Thaliana ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Actin Dynamics

Human duodenal mucosal brush border Na+/H+ exchangers NHE2 and NHE3 alter net bicarbonate movement

2001 ◽  
Vol 281 (1) ◽  
pp. G159-G163 ◽  
Author(s):  
Maltin Repishti ◽  
Daniel L. Hogan ◽  
Vijaya Pratha ◽  
Laura Davydova ◽  
Mark Donowitz ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Brush Border ◽  
Polyclonal Antibodies ◽  
Duodenal Mucosa ◽  
Transport Processes ◽  
Apical Surface ◽  
Luminal Perfusion ◽  
Significant Step

The proximal duodenal mucosa secretes HCO[Formula: see text] that serves to protect the epithelium from injury. In isolated human duodenal enterocytes in vitro, multiple luminal membrane proteins are involved in acid/base transport. We postulated that one or more isoforms of the Na+/H+ exchanger (NHE) family is located on the apical surface of human duodenal mucosal epithelial cells and thereby contributes to duodenal mucosal HCO[Formula: see text] transport. Duodenal biopsies were obtained from human volunteers, and the presence of NHE2 and NHE3 was determined by using previously characterized polyclonal antibodies (Ab 597 for NHE2 and Ab 1381 for NHE3). In addition, proximal duodenal mucosal HCO[Formula: see text] transport was measured in humans in vivo in response to luminal perfusion of graded doses of amiloride; 10−5–10−4 M amiloride was used to inhibit NHE2 and 10−3 M amiloride to inhibit NHE3. Both NHE2 and NHE3 were localized principally to the brush border of duodenal villus cells. Sequential doses of amiloride resulted in significant, step-wise increases in net duodenal HCO[Formula: see text] output. Inhibition of NHE2 with 10−5 M and 10−4 M amiloride significantly increased net HCO[Formula: see text] output. Moreover, there was an additional, equivalent increase ( P < 0.05) in duodenal HCO[Formula: see text] output with 10−3 M amiloride, which inhibited NHE3. We conclude that 1) NHE2 and NHE3 are localized principally to the brush border of human duodenal villus epithelial cells; 2) sequential inhibition of NHE2 and NHE3 isoforms resulted in step-wise increases in net HCO[Formula: see text]output; 3) NHE2 and NHE3 participate in human duodenal villus cell HCO[Formula: see text] transport; and 4) the contribution of NHE-related transport events should be considered when studying duodenal HCO[Formula: see text] transport processes.

Live Cell Imaging of Actin Dynamics in the Filamentous Fungus Aspergillus nidulans

2016 ◽  
Vol 22 (2) ◽  
pp. 264-274 ◽  
Author(s):  
Zachary Schultzhaus ◽  
Laura Quintanilla ◽  
Angelyn Hilton ◽  
Brian D. Shaw
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungus ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Tip Growth ◽  
Cell Types ◽  
Actin Dynamics ◽  
Growth Site ◽  
The Face ◽  
Over Time

AbstractHyphal cells of filamentous fungi grow at their tips in a method analogous to pollen tube and root hair elongation. This process, generally referred to as tip growth, requires precise regulation of the actin cytoskeleton, and characterizing the various actin structures in these cell types is currently an active area of research. Here, the actin marker Lifeact was used to document actin dynamics in the filamentous fungus Aspergillus nidulans. Contractile double rings were observed at septa, and annular clusters of puncta were seen subtending growing hyphal tips, corresponding to the well-characterized subapical endocytic collar. However, Lifeact also revealed two additional structures. One, an apical array, was dynamic on the face opposite the tip, while a subapical web was dynamic on the apical face and was located several microns behind the growth site. Each was observed turning into the other over time, implying that they could represent different localizations of the same structure, although hyphae with a subapical web grew faster than those exhibiting an apical array. The subapical web has not been documented in any filamentous fungus to date, and is separate from the networks of F-actin seen in other tip-growing organisms surrounding septa or stationary along the plasmalemma.

scholarly journals F-Actin Dynamics in Neurospora crassa

2010 ◽  
Vol 9 (4) ◽  
pp. 547-557 ◽  
Author(s):  
Adokiye Berepiki ◽  
Alexander Lichius ◽  
Jun-Ya Shoji ◽  
Jens Tilsner ◽  
Nick D. Read
Keyword(s):  
Neurospora Crassa ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Content Type ◽  
Actin Cables ◽  
Germ Tubes

ABSTRACT This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.

scholarly journals Similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized Madin–Darby canine kidney cells

2013 ◽  
Vol 24 (8) ◽  
pp. 1196-1207 ◽  
Author(s):  
Steeve Boulant ◽  
Megan Stanifer ◽  
Comert Kural ◽  
David K. Cureton ◽  
Ramiro Massol ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Endocytic Pathway ◽  
Cell Entry ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin–Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.

scholarly journals STUDIES ON THE ORGANIZATION OF THE BRUSH BORDER IN INTESTINAL EPITHELIAL CELLS

1965 ◽  
Vol 26 (3) ◽  
pp. 687-691 ◽  
Author(s):  
Alexander Eichholz ◽  
Robert K. Crane
Keyword(s):  
Epithelial Cells ◽  
Brush Border ◽  
Intestinal Epithelial Cells ◽  
Companion Paper ◽  
Density Gradients ◽  
Intestinal Epithelial ◽  
Fibrillar Material ◽  
Brush Borders ◽  
Individual Bands ◽  
Positively Charged

Brush borders isolated from the epithelial cells of hamster jejunum have been dissociated by treatment with 1 M Tris(hydroxymethyl)aminomethane into several subfractions which can be separated by means of centrifugation on glycerol density gradients. Investigation of the chemical specificity of disrupting agents suggests that the amino group of Tris, in its positively charged state, is involved. Five individual bands or fractions have been routinely recovered from density gradients. The distribution of alkaline phosphatase and maltase activities among these fractions has been studied and the results indicate that both enzymes are predominantly associated with one fraction which has been identified in a companion paper as being composed of the membranes of the brush border microvilli. A fibrillar material of unidentified origin has also been obtained from Tris-disrupted brush borders.

scholarly journals Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Meghan C. Drummond ◽  
Melanie Barzik ◽  
Jonathan E. Bird ◽  
Duan-Sun Zhang ◽  
Claude P. Lechene ◽  
...  
Keyword(s):  
Inner Ear ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Actin Dynamics ◽  
Length Regulation
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