Faculty Opinions recommendation of Direct laser manipulation reveals the mechanics of cell contacts in vivo.

AbstractIn the homeostatic liver, ductal cells intermingle with a microenvironment of endothelial and mesenchymal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury to replenish any lost epithelium. We have shown that liver ductal cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the biliary tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established following a microfluidic co-encapsulation that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings underscore how the relative abundance of cell-cell contacts between the epithelium and its mesenchymal microenvironment are key regulatory cues involved in the control of tissue regeneration.SummaryIn the homeostatic liver, the ductal epithelium intermingles with a microenvironment of stromal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury. We have shown that these cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the portal tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal niche cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established through a microfluidic co-encapsulation method that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings re-evaluate the concept of the cellular niche, whereby the proportions of cell-cell contacts between the epithelium and its mesenchymal niche, and not the absolute cell numbers, are the key regulatory cues involved in the control of tissue regeneration.

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3D-microdevice for the in vivo capture of cancer-associated circulating cells.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.6_suppl.e579 ◽

2017 ◽

Vol 35 (6_suppl) ◽

pp. e579-e579

Author(s):

Hélène Cayron ◽

Alejandro Kayum Jiménez Zenteno ◽

Aurore Esteve ◽

Sylvain Sanson ◽

Christophe Vieu ◽

...

Keyword(s):

Cancer Cells ◽

Expression Profiles ◽

Antigen Expression ◽

Sampling Bias ◽

Additional Treatment ◽

In Vitro Assays ◽

Major Drawback ◽

Direct Laser

e579 Background: Circulating tumor cells (CTCs) are cancer cells that have detached from a tumor and have entered into the blood circulation at a very low concentration (D. Shook, Mech. Dev., Nov 2003). CTCs have a strong prognostic value, as their number has been correlated to overall survival in different metastatic cancers (J. S. de Bono, Clin. Cancer Res., Oct 2008). Considering the rareness of CTCs in blood, capturing them in vitro is very challenging. CTCs being mainly larger and less deformable than most of blood cells, ISET was the first system exploiting their physical traits using a filtration membrane to enrich 10mL blood samples (G. Vona, Am. J. Pathol., Jan 2000). However, placing the trapping system directly within the bloodstream would increase the amount of blood screened and ensure no sampling bias. To our knowledge, the only system developed for in vivo capture of CTCs relies on an immunologic detection targeting CTCs with specific epithelial-cell adhesion molecules (N. Saucedo-Zeni, Int. J. Oncol., Oct 2012). The major drawback of this technique is the selection bias induced, given the strong heterogeneity of antigen expression profiles in CTC population as confirmed by several studies. Methods: Our device combines the advantages of in vivo capture and physical trapping of CTCs. A polymeric 3D net-like microdevice is fabricated using a Direct Laser Writing technique (Nanoscribe) and integrated onto a Nitinol guidewire to be introduced into the basilic vein through a routine 20G catheter. To optimize the design, we conducted simulation studies and in vitro assays using a fluidic platform reproducing in vivo conditions. Results: We succeeded in capturing PC3 human prostate cancer cells from 20 mL healthy donor blood spiked with 1,000 PC3 cells in 2 minutes, demonstrating the capability to capture CTCs in conditions close to those found in vivo, in terms of pressure and flow rate and without any additional treatment or dilution of the blood. Conclusions: This device could facilitate treatment personalization and follow-up. Its versatility should render it transposable to the capture of single or clustered CTCs, derived from all types of cancer and, by extension, to other circulating cellular and molecular biomarkers.

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Direct laser manipulation reveals the mechanics of cell contacts in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418732112 ◽

2015 ◽

Vol 112 (5) ◽

pp. 1416-1421 ◽

Cited By ~ 123

Author(s):

Kapil Bambardekar ◽

Raphaël Clément ◽

Olivier Blanc ◽

Claire Chardès ◽

Pierre-François Lenne

Keyword(s):

Cell Shape ◽

Constitutive Law ◽

Cell Junctions ◽

Scale Model ◽

Tissue Morphogenesis ◽

Cell Contacts ◽

Cell Shape Changes ◽

Shape Changes ◽

Cell Cell

Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell–cell and cell–ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell–cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.

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Continual assembly of half-desmosomal structures in the absence of cell contacts and their frustrated endocytosis: a coordinated Sisyphus cycle.

The Journal of Cell Biology ◽

10.1083/jcb.131.3.745 ◽

1995 ◽

Vol 131 (3) ◽

pp. 745-760 ◽

Cited By ~ 74

Author(s):

M P Demlehner ◽

S Schäfer ◽

C Grund ◽

W W Franke

Keyword(s):

Adjacent Cell ◽

Cell Contact ◽

Membrane Domains ◽

Cell Contacts ◽

Desmosomal Cadherins ◽

L Cells ◽

Transmembrane Glycoprotein ◽

Cell Structures ◽

Cytoplasmic Vesicles

It is widely assumed that the coordinate assembly of desmosomal cadherins and plaque proteins into desmosome-typical plaque-coated membrane domains, capable of anchoring intermediate-sized filaments (IF), requires cell-to-cell contacts and a critical extracellular Ca2+ concentration. To test this hypothesis we studied several cell lines grown for years in media with less than 0.1 mM Ca2+ to steady-state low Ca2+ medium (LCM) conditions, particularly the human keratinocyte line HaCaT devoid of any junctional cell contact (HaCaT-L cells). Using immunolocalization and vesicle fractionation techniques, we found that the transmembrane glycoprotein, desmoglein (Dsg), colocalized with the plaque proteins, desmoplakin and plakoglobin. The sites of coassembly of desmosomal molecules in HaCaT-L cells as well as in HaCaT cells directly brought into LCM were identified as asymmetric plaque-coated plasma membrane domains (half-desmosomes) or as special plaque-associated cytoplasmic vesicles, most of which had formed endocytotically. The surface exposure of Dsg in these half-desmosomes was demonstrated by the binding, in vivo, of antibodies specific for an extracellular Dsg segment which also could cross-bridge them into symmetric quasi-desmosomes. Otherwise, these half-desmosomes were shown in LCM to be taken up endocytotically. Half-desmosomal assemblies were also seen in uncoupled cells in normal Ca2+ medium. We conclude that, in the absence of intercellular contacts, assembly of desmosomal proteins at the cell surface takes place, resulting in transient half-desmosomes which then, in LCM and without a stable partner connection to the adjacent cell, can be endocytotically resumed. This frustrated cycle of synthesis and assembly maintains an ensemble of molecules characteristic of epithelial differentiation and the potential to form desmosomes, even when the final junctional structure cannot be formed. We propose that these half-desmosomal structures are general cell structures of epithelial and other desmosome-forming cells.

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Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro

Reproduction ◽

10.1530/rep-06-0385 ◽

2007 ◽

Vol 133 (6) ◽

pp. 1169-1179 ◽

Cited By ~ 97

Author(s):

Tu’uhevaha J Kaitu’u-Lino ◽

Pavel Sluka ◽

Caroline F H Foo ◽

Peter G Stanton

Keyword(s):

Mrna Expression ◽

Sertoli Cell ◽

Sertoli Cells ◽

Hormonal Regulation ◽

Cell Contacts ◽

Barrier Formation ◽

Protein Components ◽

Blood Testis Barrier

Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood–testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJsin vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P< 0.01) increased TER twofold and claudin-11 mRNA two- to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive intercellular contacts. In contrast to claudin-11, Tand FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesisin vivois partly via their effects on TJ proteins and regulation of the blood–testis barrier.

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Development of desmosomal adhesion between MDCK cells following calcium switching

Journal of Cell Science ◽

10.1242/jcs.97.4.689 ◽

1990 ◽

Vol 97 (4) ◽

pp. 689-704

Author(s):

D.L. Mattey ◽

G. Burdge ◽

D.R. Garrod

Keyword(s):

Protein Synthesis ◽

Cell Surface ◽

Mdck Cells ◽

Surface Concentration ◽

Cell Contacts ◽

Gradual Development ◽

Intercellular Adhesions ◽

Initial Assembly

The development or maturation of intercellular adhesions following their initiation has received very little attention even though this is an area of significance for a variety of in vivo processes. Using Ca2(+)-induced desmosome formation in MDCK cells as a study system it is shown that, following its initiation, desmosome formation continues for many hours. Following Ca2+ switching the major desmosomal glycoproteins, dg2/3a,b (desmocollins), accumulate progressively at the cell surface. Accumulation is first detectable within 45 min, but continues linearly for approximately 16 h, reaching a plateau at 24–32 h at 15 times the amount present in low-Ca2+ medium (LCM). Desmosomes do not increase in size during this time, but appear to become more numerous. These results suggest that cells progressively increase their desmosome-mediated adhesion over this period of time. Cycloheximide treatment shows that approximately 93% of the total dg2/3a,b accumulation is dependent upon protein synthesis after Ca2+ switching and only approximately 7% on assembly of pre-synthesised material. Thus, although triggering of desmosome formation is rapid, protein synthesis makes a major contribution to the gradual development of desmosomal adhesion in these cells. The initial assembly phase itself can be inhibited by treating cells in LCM with chloroquine, which reduces the cell surface concentration of dg2/3a,b by 40–50%. However, slow dg2/3a,b accumulation does take place in chloroquine and, if protein synthesis is permitted, desmosome formation occurs. It is suggested that when cell contacts are formed in vivo, maximisation of intercellular adhesiveness may take many hours and is dependent on the synthesis and accumulation of adhesive components.

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Interactions between Myeloma Cells and Osteoclasts Are Controlled by Unique Histological Structures Involving Vascular Compartments.

Blood ◽

10.1182/blood.v106.11.2513.2513 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2513-2513

Author(s):

Thomas L. Andersen ◽

Katarzyna E. Skorzynska ◽

Teis E. Sondergaard ◽

Trine Plesner ◽

Ellen Hauge ◽

...

Keyword(s):

Bone Marrow ◽

Cell Wall ◽

Vascular Remodeling ◽

Type Iii Collagen ◽

Bone Marrow Biopsies ◽

Myeloma Cells ◽

Cell Contacts ◽

Specialized Cell ◽

Direct Cell

Abstract Although myeloma development and enhanced bone resorption are intimately related, the mechanism responsible of this relation is not known. In vitro studies have stressed the critical role of direct cell contacts between myeloma cells and osteoclast. In vivo, however, little is known about the organization of the cells present at osteolytic lesions, because of the complexity of cell-cell interactions in the bone marrow of myeloma patients. Therefore, we conducted an immunohistochemical study with multiple stainings allowing the simultaneous identification of different cell types at resorption sites of bone marrow biopsies of myeloma patients. The biopsies showed that in average 1% of the bone surface was lined by mature multinucleated TRAP+ osteoclasts, but that only 6% of these osteoclasts showed direct contacts with myeloma cells. The biopsies showed also TRAP+ mononucleated pre-osteoclasts in the bone marrow compartment, and 40% of these pre-osteoclasts showed direct contacts with myeloma cells. Bone marrow pre-osteoclasts show thus much more frequent contacts with myeloma cells, compared with mature osteoclasts lining bone surfaces. These respective values remained unchanged, whether the myeloma cells were identified through CD138 or through light chain expression (counts in biopsies from 13 patients). Importantly, we found that 80% of the osteoclasts lining the bone surfaces, were separated from the bone marrow compartment by a specialized cell wall (seen in all biopsies of the 15 patients, that were analyzed). This wall consists of a single layer of (sometimes very) flattened cells lining the bone marrow, and expressing NCAM, propeptide of type III collagen, and osteocalcin, but not CD34. When performing 3D reconstructions by using serial sections, this wall appeared as a continuous roof covering the bone surfaces undergoing remodeling, and connected to the bone lining cells at its periphery. Furthermore, CD34 staining revealed that capillaries are abundant at the bone marrow side of this cell wall, and that some show an opening into the wall. These capillaries may thus allow communication between the bone marrow and the bone surfaces undergoing remodeling, and render the bone remodeling compartments vascular-like. In addition, the TRAP+ preosteoclast detected in the bone marrow space, were positioned along the capillaries leading to the vascular/remodeling compartments. In conclusion, this study shows that in vivo, interactions between myeloma cells and osteoclasts are mediated only rarely through direct cell contacts, and identifies for the first time unique cell arrangements that are likely to play a role in these interactions: a specialized cell wall separates the bone marrow from the vascular/remodeling compartments in most resorption sites (80% of the osteoclasts), and the cells of this wall are thus in a privileged situation to control myeloma-osteoclast interactions; capillaries connect the marrow cavity with the vascular/remodeling compartment, thereby allowing guidance of pre-osteoclasts from the bone marrow to the resorption sites; the generation of these pre-osteoclasts may be stimulated by the high incidence of their direct contacts with myeloma cells in the bone marrow.

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