Conceived and designed the experiments: AKN KJ. Performed the experiments: AKN. Analyzed the data: AKN KJ. Wrote the paper: AKN KJ.
The authors have declared that no competing interests exist.
Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the “fungipod”, formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7±5.6 µm long and 1.8±0.67 µm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225±55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen
Yeasts are normal microbial commensals of humans and a significant source of opportunistic infections, especially in immunocompromised individuals. We report a novel cellular protrusive structure, the fungipod, which participates in the host-microbe interaction between human immature dendritic cells (DC) and yeasts. The fungipod's structure is based on and propelled by a robust process of local actin cytoskeleton growth at the DC-yeast contact site, and this cytoskeletal remodeling results in a durable tubular structure over 10 µm long connecting the dorsal DC membrane and yeast. The fungal cell wall polysaccharides mannan and chitin trigger fungipod formation by stimulating the carbohydrate pattern recognition receptor CD206. Fungipods are part of a specific response to large particulate objects (i.e., yeast), and they may promote the human immature DC's relatively poor phagocytosis of yeast. The human fungal pathogen,
The innate immune response against fungal pathogens is effected by macrophages, neutrophils and dendritic cells (DC). DC may encounter opportunistic fungi, such as
Infection by
The fungal cell wall contains mannan, β-glucans, chitin and other carbohydrates recognizable as non-self epitopes
Internalization is central to many aspects of CD206 biology. Indeed, the majority of CD206 is found within the endocytic system, and internalization of antigen via CD206 in DC results in MHC class II antigen presentation for T cell activation
Dendritic actin network polymerization via Arp2/3 is a recurrent theme in host cell-microbe interactions. Arp2/3 complex binds to existing F-actin and upon activation nucleates new filament growth from this branch point
Activation of the actin remodeling machinery is a normal part of the professional phagocyte's response to microbes (although it is sometimes co-opted by pathogens). This machinery must be directed by pattern recognition receptors to recognize invading microbes. In this report we describe a novel actin-based protrusive structure formed by DC in response to ligation of CD206 by yeast cell walls.
We found that human monocyte-derived DC generated peculiar dorsal pseudopodial structures after several hours of exposure to zymosan. We designated these protrusions “fungipods”. They were visible in DIC imaging, resembling long tethers connecting DC and zymosan, and were comprised of an apparently smooth, well-ordered distal region tapering into a convoluted cell-proximal region. Distal fungipods averaged 7.4±3.3 µm long (N = 35, range: 2.7–16.8 µm) and 1.8±0.67 µm wide (range: 0.92–4.6 µm) at the contact site. Their overall length was 13.7±5.6 µm (range: 5.9–25.3 µm) (
(A) DIC images of representative fungipods on the dorsal surfaces of immature DC after 4 hours exposure to zymosan. “Z” denotes the location of the fungipod attached zymosan particle. Arrows and arrowheads designate distal and proximal fungipod regions, respectively. Bars = 10 µm. (B) Schematic of fungipod morphology and distribution of measured contact site widths as well as lengths of distal, proximal and total fungipod regions (N = 35). (C) SEM image of a typical fungipod with roughly cylindrical distal geometry (9500x). Arrowhead denotes the attached zymosan particle. Bar = 1 µm. (D) SEM image of a fungipod with ribbon-like distal geometry displaying longitudinal ridges. Right panel shows a higher magnification view of the flattened structure in the distal fungipod from the bracketed area of the left panel. Left panel, 7500x; Right panel, 25000x, Bar = 1 µm. (E) DiI-labeled membrane of a distal fungipod at the zymosan contact site (arrow) viewed as a medial confocal section of the distal fungipod. (left, DIC; right, confocal fluorescence). Bars = 5 µm. (F) A fungipod/zymosan contact site seen in thin section TEM imaging. The right panel displays the left panel bracketed region at higher magnification. Designations: “Z”, Zymosan; “*”, distal fungipod; arrow, example of vesicle inside distal fungipod; arrowheads, examples of pits/membrane densities at the contact site. Left panel, 2700x; Right panel, 6500x; Bars = 500 nm. (G) Thin section TEM of membrane invaginations at a zymosan contact site displaying studded juxtamembrane densities. 11000x, Bar = 500 nm. (H) DIC imaging of the initial attachment and nascence of a fungipod. Arrowheads and arrows designate a zymosan particle associated with fungipod generation and the nascent fungipod, respectively. Times indicate elapsed time of DC attachment for the indicated zymosan particle. Bar = 10 µm. (I) DIC imaging of the maturation of a fungipod. Arrowheads and arrows designate a relevant zymosan particle and the growing fungipod, respectively. Times indicate elapsed time since the advent of the fungipod. Bar = 10 µm. (J) DIC imaging of the growth of a mature fungipod. Bar = 10 µm. (K) DIC imaging of fungipods (arrowheads) associated with zymosan and DC for several hours. Bar = 10 µm. (L) DIC imaging of a fungipod (arrowhead) formed by a DC in response to live
Fungipodial structures required association of zymosan particles with the plasma membrane. Blocking with excess soluble mannan plus the β-glucan laminarin completely inhibits interaction of zymosan with CLR on DC
Early zymosan-induced fungipodial extensions were often thin and lacked a well-ordered distal fungipod, but they matured into the previously described fungipods over the course of approximately 45–60 minutes (
We confirmed that DC form fungipods after exposure to live cultures of budding
Finally, we also considered that fungipod formation might arise only in DC highly stimulated via the acquisition of many zymosan particles. In this dose-dependence model, fungipod formation efficiency would be positively correlated with zymosan dose such that high fungipod formation efficiencies would be predicted at high dose and vice versa (
(A) Dose-dependence of fungipods predicts higher fungipod formation efficiencies at higher degrees of DC stimulation by zymosan particles. (B) Fungipod formation efficiency in DC-zymosan cultures is independent of zymosan density applied to DC. (n.s., not significant by Student's t-test) (C) Individual DC show no positive correlation between degree of zymosan stimulation and fungipod formation efficiency. For panels B & C, N = 165 cells for 20 µg/ml zymosan and N = 145 for 2 µg/ml zymosan. (D) There is no apparent correlation between degree of DC stimulation by zymosan and the kinetics of fungipod formation (N = 39 individual fungipod formation events).
Distal fungipods contained copious F-actin in a structure tapering away from the contact site (
(A) Left panel: Red/green anaglyph of actin in DC fungipods (arrowheads) after 4 h exposure to zymosan. Non-fluorescent zymosan particles contacted the distal fungipod ends. Right panel: A single z-axis plane DIC image showing the position of zymosan particles (asterisk) with respect to fungipods (arrowhead). Bar = 10 µm. (B) Images of zymosan induced fungipods (arrowheads) in DIC (left panel) and actin-stained epifluorescence (right panel). The DIC image represents a single z-axis plane above the dorsal membrane, and actin is shown as a maximum intensity projection of all z-axis planes. Arrow indicates proximal fungipod. Bar = 10 µm. (C) DIC images of 4 hour zymosan-exposed DC fungipods before (“pre”) and after (“post”) treatment with cytochalasin D (15 minutes, 1 µm). Fungipods were well ordered before treatment (red arrowheads) and disordered after treatment (green arrowheads). Bar = 5 µm. (D) DIC (left panel) and confocal fluorescence imaging (right panel) of fungipods on DC after 4 hour zymosan exposure showing actin in red and cortactin in green at a fungipod-medial z-axis plane. Arrow and asterisk denotes fungipod and attached zymosan, respectively. Bar in left panel = 10 µm. Arrowheads in right panel designate the contact site and right panel inset is an orthogonal section through the contact site. Inset bar = 1 µm. (E) Linescan of actin (red) and cortactin (green) intensities (arbitrary units) in the distal fungipod. Location of line is shown in the inset image. (F) Confocal imaging of α-tubulin in the DC cell body showing microtubules. (G) (i) DIC image of fungipods at a medial z-axis plane with zymosan particles marked by asterisks. (ii) Diffuse α-tubulin staining in the distal fungipod. Blue line represents the region used for orthogonal section. (iii) Orthogonal section of previous panel showing fungipod diffuse tubulin in cross-section. Green and red arrowheads denote the z-axis positions of the cell body and fungipod images, respectively. Bar = 5 µm. (H) Fungipods (arrowheads) persist even after 1 hour exposure to nocodazol (10 µM). Bar = 10 µm.
We found that the actin nucleation factor cortactin was abundantly localized to the distal fungipod. Interestingly, cortactin was configured in a conical sheath with a core rich in F-actin (
We next examined the involvement of microtubules in fungipods. Immunofluorescence localization of α-tubulin revealed normal microtubular staining in the DC cell body (
As previously mentioned, elongating fungipods were often apparent in DIC time series. Upon closer examination we noted that a rearward flow of refractile material moving from the contact site toward the cell body was visible in the distal fungipod (
(A) Representative DIC image showing the zymosan particle (asterisk), contact site (arrowheads) and line used for kymograph analysis. Bar = 5 µm. (B) Representative kymograph showing DIC intensity along the line from the previous panel over time. Rearward flow appears as diagonal lines and speed of flow is measured from the slope of these lines. (C) DiI labeled distal fungipod overlaid with a line indicating the region used for kymography to observe changes in the fungipod edge position over time. Asterisk indicates position of attached zymosan. Bar = 5 µm. (D) Kymograph showing non-undulating edges of DiI stained distal fungipod. Arrowheads mark the edges of the fungipod. (E) SEM image of distal fungipods with arrows emphasizing the smooth lateral edges of these structures. Bar = 1 µm. (F) DIC time series showing rotation of a zymosan particle attached to a fungipod. The crosses and asterisks indicate the positions of the site of proximal fungipod attachment to the cell body and the zymosan particle, respectively. Bar = 10 µm. (G) DIC image of a fungipod showing a kinked appearance (arrowheads) developed during growth of the structure. Bar = 10 µm. (H) DIC time series showing supercoiling of a fungipod (arrowhead) attached to the zymosan particle indicated by an asterisk. Bar = 5 µm. (I) DIC time series showing a kinked fungipod (red arrowhead) associated with motion of the attached zymosan into the cell membrane as shown by the yellow reference line and green arrowhead. Blue arrowhead emphasizes relaxation of the kinked fungipod. Bar = 10 µm.
We also observed that fungipodial growth is accompanied by rotation about the longitudinal axis of the fungipod. In cases where the attached zymosan particle became detached from the cell surface while still bound at the fungipod contact site, the fungipod particle was observed to rotate or be driven in a circular motion (
We tested whether ligation of the phagocytic receptor Dectin-1 by zymosan was involved in the generation of fungipodial protrusions. The β-glucan laminarin binds Dectin-1 and inhibits its interaction with zymosan particles. We found that blocking with excess soluble laminarin did not inhibit the formation of zymosan-induced fungipods or number of zymosans per cell (
(A) Laminarin (5 mg/ml) blocked DC produced normal fungipods. (B) Anti-dectin-1 polyclonal antibody (10 µg/ml) blocked DC produced normal fungipods. (C) Pharmacological inhibition of Dectin-1 signaling with Syk inhibitor II (1 µM) does not block formation of normal fungipods. (D) Blocking with soluble mannan (5 mg/ml) does inhibit the formation of fungipods although DC-associated zymosan particles are still observed. (E) Internalized zymosan particles (arrowheads) are still observed in the presence of blocking with soluble mannan. (F) Mannan-coated 5 µm beads (asterisk) induced protrusions (arrow) identical to fungipods. (G) Chitin particles induced protrusions identical to fungipods. Inset contains the bracketed area reproduced at 2× higher magnification than the main panel and focused at a lower plane to allow imaging of the chitin particle. (H) Blocking with anti-CD206 polyclonal antibody (50 µg/ml) abolished formation of zymosan induced fungipods but did not prevent binding to DC or internalization. (I) Anti-CD206 antibody coated 5 µm beads induced the formation of protrusions identical to fungipods. (J) DIC (left panel) and epifluorescence (right panel) imaging of intense CD206 staining at zymosan-DC contact sites (arrowheads). (K) DIC (left panel) and confocal fluorescence (right panel) imaging of clathrin light chain in zymosan (asterisks) induced fungipods. Right panel is a maximum intensity projection in the z-axis of a three dimensional confocal stack. Arrows indicate fungipods bearing clathrin LC. (L) DIC (left panel) and confocal fluorescence (middle and right panels) imaging of dynamin in DC/zymosan (asterisks) contacts. Lines denote locations of orthogonal sections through a membrane ruffle near a zymosan particle (arrowhead) and a mature fungipod (arrow). Orthogonal sections are shown in the right panels. Bar = 10 µm.
Treatment with excess soluble mannan completely inhibited formation of fungipodial protrusions (
We adsorbed mannan to 5 µm polystyrene beads and exposed DC to these beads. We found that bead-immobilized mannan was sufficient to generate fungipods with similar size and structure to those observed after zymosan exposure (
Blocking CD206 with anti-CD206 polyclonal antibody prior to addition of zymosan dramatically inhibited formation of fungipods and the efficiency of their formation (
CD206 was greatly enriched at most zymosan/DC contact sites including the contact site on the distal fungipod (
We have noted that fungipod/yeast attachments appear quite durable as we do not observe loss of yeast particles over hours of imaging and the contacts are quite tightly apposed. Furthermore, we often saw single zymosan particles contacted by multiple distinct fungipods (as many as 14) (
(A) DIC (left panel) and confocal 3D projection (right panel, red/green anaglyph) images of actin staining in DC with a zymosan particle attached in a cup-like structure formed by 14 fungipods. Zymosan position is shown in DIC. Bar = 10 µm. (B) SEM image (3700x) of zymosan particles with numerous attached fungipods. Bar = 10 µm. (C) DIC (left panel), confocal fluorescence (middle panel), and confocal 3D projection (right panel, red/green anaglyph) images of DiI stained DC with fungipods (arrows) enmeshing a zymosan particle (asterisk). Bar = 10 µm. (D) TEM thin section image of a zymosan particle (asterisk) being monolaterally engulfed (arrowheads) by a DC. Bar = 1 µm. (E) TEM thin section image of an internalized zymosan particle (asterisk) associated with overlapping phagocytic membranes (arrowheads). Bar = 1 µm. (F) TEM thin section image of a zymosan particle (asterisk) being bilaterally engulfed (arrowheads) by a DC. Bar = 1 µm. (G) DIC (i) and confocal fluorescence (ii) images of zymosan (asterisk) associated membrane wedges on DC (arrowheads) stained for actin (red) and cortactin (green). Orthogonal sections of a membrane wedge through horizontal (purple) and vertical (cyan) lines shown are depicted in panels (iii) and (iv), respectively. Bar = 10 µm for panels (i, ii). Bar = 1 µm for panels (iii, iv). (H) Representative DIC image of anti-CD206 coated 1 µm beads (arrowheads) bound to DC for 4 hours resulting in no fungipod-like protrusions. Bar = 10 µm.
To examine the kinetics and probability distribution of internalization and/or fungipod formation, we have undertaken an extensive quantitative analysis of individual zymosan histories (N = 301) as observed in their interactions with DC during a cumulative ∼980 hours in contact with DC. We quantified the distribution of zymosan in various states (DC surface bound, fungipod-associated, and internalized) as well as the kinetics of transition between those states (summarized schematically in
Coiling phagocytosis has been described in the internalization of
While anti-CD206 coated 5 µm beads do generate fungipods, 1 µm beads coated at the same surface density of anti-CD206 are not capable of generating fungipodial protrusions despite avid binding to the DC membrane (
We co-cultured DC with log-phase live
(A) Percent of DC possessing surface-bound live yeast particles that made fungipods after 4 hour, 37°C DC co-culture with
We have described a novel protrusion, termed the fungipod, that is produced on immature DC by fixed and live
The distal fungipod's close contact site with the zymosan particle often contained regions of greater electron density along the apposed membrane visible in TEM thin sections (
The cytoplasmic domain of CD206 contains a tyrosine-based internalization motif responsible for mediating endocytosis via clathrin coated pits
Dynamin can recruit cortactin to the membrane leading to actin polymerization. In addition to this localization, cortactin activity can be regulated by Src-family kinases (SFK)
Consistent with the hypothesis that CD206 concentration at zymosan contact sites catalyzes the development of clathrin patches leading to dynamin recruitment, we have observed dynamin enrichment in small membrane protrusions next to zymosan particles. Since dynamin binds cortactin
(A) Hypothetical protein complex involved in initiation and growth of fungipods. CD206 recruits clathrin complexes via AP-2 interactions with its tyrosine-based internalization motif. Clathrin binds dynamin, which can recruit cortactin leading to stabilization of Arp2/3 complexes and elongation of F-actin in dendritic networks. Cortactin also stabilizes actin branch points permitting greater longevity and stiffness of the F-actin network. Steady state polymerization of actin at the zymosan-proximal tip results in rearward flow of actin and a reaction force pushing the zymosan particle outward. (B) Summary of potential functional significances of fungipods for DC biology. (1) Fungipods may improve binding and retention of large particles such as zymosan that would be easily removed by shearing contact with other cells. (2) Fungipods may assist in phagocytosis (including monolateral engulfment/coiling phagocytosis) of yeast particles by holding this large particle in place during engulfment. (3) Fungipods may be part of the DC's specific size discriminatory response program directed against large (i.e., 5 µm) particles, but not smaller particles (i.e., 1 µm) that are more easily dealt with through conventional phagocytic means.
Heinsbroek, et al have observed a sequential engagement of C-type lectins by zymosan and
Several features of the zymosan-induced fungipods described in this report bear similarity to actin comet tails associated with
We asked whether our observations of growth in the distal fungipod are comparable to what is understood about actin polymerization and actin rocketing systems in vivo and in vitro. Our measurements of rearward flow in the distal fungipod imply a dendritic actin growth speed of ∼200 nm/second. Assuming actin growth at the distal tip in a 1 µm diameter circular contact with filament barbed ends placed at 40 nm intervals
What could be the functional relevance of fungipods to DC function and microbe internalization in general? On the basis of our observations, we propose the following areas of functional significance: 1) Improved binding/retention of particles, 2) Promotion of phagocytosis (including coiling phagocytosis), 3) Participation in a size discrimination program in phagocytes (
Fungipodial protrusions may promote phagocytosis of yeasts by improving long-term retention. DC experience long contact times with zymosan particles prior to internalization, and many bound particles are not phagocytosed despite hours of contact (
Previous reports have identified “coiling phagocytosis”, a monolateral engulfment that acts to internalize bacteria and yeast via a single pseudopod that wraps around the particle and creates a phagosome. We have identified examples of coiling phagocytosis occurring under conditions where fungipods are present (
Professional phagocytes such as macrophages and immature DC must be able to recognize and respond to pathogens with a wide range of sizes from viruses of <100 nm diameter to much larger extracellular pathogens (i.e., helminthes, filamentous fungi and
We found that yeast-sized mannan or anti-CD206 coated beads (5 µm) induced fungipod formation while similar particles of 1 µm diameter did not suggesting that fungipods are part of a size discrimination capability of immature DC. According to our model of fungipod formation (
Pathogenic fungi, such as
We observed that DC interaction with
PBMC were isolated from human peripheral blood buffy coats purchased from New York Blood Center (New York, NY). Monocytes were isolated by adherence on tissue culture treated plastic flasks. Immature dendritic cells were prepared by culturing monocytes with 500 U/ml human IL-4 and 800 U/ml human GM-CSF (Peprotech, Rocky Hill, NJ) in RPMI-1640 medium with 10% heat inactivated FBS in glass-bottom MatTek dishes (MatTek Corp., Ashland, MA) for 6 days. Immature macrophages were produced via the same procedure but with omission of IL-4. DC and macrophages were activated with 250 ng/ml LPS (Sigma, St. Louis, MO) for 24 hours prior to use.
Unlabeled, formalin killed zymosan was obtained from Invitrogen (Carlsbad, CA) and resuspended as a PBS stock. Zymosan was used at a final concentration 20 µg/ml (unless otherwise noted) after 3×15 second vortexing (max speed) and 3×15 second bath sonication to achieve monodispersion. Cells were incubated with zymosan for 4 hours, 37°C unless otherwise noted. Live, wild-type
Samples prepared for all light microscopic observations except CD206 staining were fixed 20 minutes with 37°C 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS, pH 7. This fixation provided optimal preservation of fungipod structures, but was not compatible with CD206 immunostaining. For CD206 fluorescence imaging, samples were fixed 20 minutes with 37°C 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS, pH 7.
Wide field light microscopy was performed on an Olympus IX81 inverted microscope with a 60x, 1.4 NA oil objective lens, stage with z-axis stepper motor control, and objective-based autofocus (Olympus, Center Valley, PA). A 37°C, CO2 controlled stage insert (Warner Instruments, Hamden, CT) was used for live cell imaging. DIC images, except those accompanying confocal fluorescence images, were taken using the DIC optical train of this microscope. A 100 W Hg arc lamp provided epifluorescence illumination. Filters and dichroic mirrors (Chroma, Rockingham, VT) for Alexafluor-488 imaging were as follows: excitation, 488/10; emission, 535/25; dichroic, 475/25. For DiI imaging we used the following: excitation, 535/50; emission, 605/40; dichroic, 530/20. Images were captured using an air-cooled SensiCam QE CCD camera (Cooke Corp., Romulus, MI) driven by Metamorph (Molecular Devices, Downingtown, PA).
Laser scanning confocal microscopy was performed on a Zeiss 510 Meta inverted instrument using a 63x, 1.4 NA oil objective lens. Samples were illuminated with 488 nm and 543 nm lines from a 30 mW Ar ion laser and a 1 mW He-Ne laser, respectively. We used a UV/488/543/633 main dichroic mirror and a NFT545 secondary dichroic in cases of dual color imaging. Alexafluor-488 emission was collected using a LP505 emission filter, and rhodamine fluorescence was collected with a LP560 filter. Data were collected in 1024×1024 pixel format and 12-bit depth, with non-interlaced, descanned, multitracked scanning and 4 scan averaging. Z-axis steps were taken in increments of 200 nm.
Primary antibodies used were as follows: cortactin (4F11; Millipore, Temecula, CA), clathrin light chain (CON.1; Santa Cruz Biotechnology, Santa Cruz, CA), CD206 (“anti-hMMR”; R&D Systems, Minneapolis, MN). Antibody staining was done with 10 µg/ml primary antibody, 30 minutes, 25°C. Secondary antibodies (anti-mouse or goat IgG, as appropriate) labeled with Alexafluor-488 (Invitrogen, Carlsbad, CA) were used at 1 µg/ml, 30 minutes, 25°C. F-actin was stained with rhodamine-phalloidin (Invitrogen) according to the manufacturer's instructions. Membrane staining with DiI-C18 (Sigma, St. Louis, MO) was done at 1 µg/ml.
Samples were prepared by fixation in 2.5% glutaraldehyde in 0.1 M Cacodylate (pH 7.3) followed by 1% OsO4/Cacodylate, dehydration in graded ethanol, critical point drying (CPD 030; Bal-Tec, Vienna, Austria), and sputter coating (Polaron E-5100; Quorum Technologies; East Sussex, UK). Observations were performed using a JEOL 6300 SEM with an Orion Digital Micrography System.
Samples were prepared by fixation in 2.5% glutaraldehyde, 1% tannic acid, 0.1 M Cacodylate (pH 7.3), stained with 2% Uranyl acetate, dehydrated in graded ethanol, and embedded in Epon. 60 nm sections were cut and stained with 2% Uranyl acetate followed by Sato lead stain. Observations were performed using a Technai 12 TEM with a Gatan Multiscan 794 digital camera.
Blocking reagents were used at the following concentrations: Mannan (10 mg/ml; Sigma, St. Louis, MO), Laminarin (5 mg/ml; Sigma), anti-Dectin-1 polyclonal antibody (10 µg/ml; “anti-hdectin-1/CLEC7A”, R&D Systems, Minneapolis, MN), anti-CD206 polyclonal antibody (50 µg/ml; “anti-hMMR”, R&D Systems). Syk Inhibitor II (EMD Biosciences, Gibbstown, NJ) was used at 1 µM. For blocking and inhibitor experiments, cells were exposed to the agent at 30 minutes, 37°C prior to the addition of zymosan. However, cytochalasin D and nocodazol were used acutely at 1 µM.
Nominal 5 µm (4.58±0.07 µm) and 1 µm (1.053±0.01 µm) polystyrene beads were obtained from Polysciences (Warrington, PA). Ligands were passively adsorbed on beads using equivalent total surface area of beads in all reactions. After washing with 50 mM bicarbonate buffer (pH 9), adsorption was done in 100 µl total volume (same buffer) at 25°C overnight. For mannan and laminarin, the adsorption reaction concentration was 10 mg/ml. For anti-CD206 polyclonal antibody (R&D systems, Minneapolis, MN) the concentration was 50 µg/ml. For negative controls, beads were coated in 1 mg/ml bovine serum albumin (Sigma, St. Louis, MO), 1 mg/ml chicken egg ovalbumin (Sigma) or neat fetal bovine serum. Beads were washed in PBS and used immediately. Chitin particles (1–10 µm) were prepared by probe sonication and centrifugation as previously described
UniprotKB accession numbers for human proteins referenced in these data are as follows: DC-SIGN (CD209), A8MVQ9; Mannose Receptor (MRC1, CD206), P22897; Dectin-1 (CLEC7A), Q9BXN2; Cortactin, Q96H99; Syk, P43405; Clathrin Light Chain A & B, P09496 & P09497; Dynamin 1 & 2, Q05193 & P50570.
Macrophage-produced fungipods and LPS-induced diminution of fungipod formation efficiencies. (A) A human monocyte derived immature macrophage was treated 4 hours with zymosan and fixed. Arrowheads denote zymosans associated with fungipods on a macrophage. Yellow line indicates cell boundary (below focal plane) and bar = 10 µm. (B) Fungipod formation efficiency was calculated for 4 hour zymosan exposed human monocyte derived immature dendritic cells (DC, circles) and immature macrophages (Mac, triangles) untreated (black symbols) or activated with LPS for 24 h prior to zymosan exposure (red symbols). Bars denote average values and statistical significance between untreated and LPS-activated cells (Student's t-test) is provided below the graph. Efficiency was calculated as the number of fungipods per cell divided by number of plasma membrane bound zymosans per cell.
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Summary of blocking experiment results. Dendritic Cells were exposed to zymosan for 4 hours either with no treatment or with various blocking reagents as described in
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Quantitative analysis of zymosan interaction with dendritic cells with respect to fungipod formation and phagocytosis. (A) Schematic representation of possible zymosan states (“S”, surface bound zymosan; “F”, fungipod associated zymosan; “I”, internalized zymosan) and transitions between states (color coded as shown) that were considered in this analysis. (B) Observed kinetics of state transitions color coded as above. Triangles represent data points where the entire transition was not observable (i.e., zymosan bound prior to start of time lapse, so the exact time of binding was not observed), and thus these are observed minimal times of transition. Circles indicate data points where the entire transition was observed. This data was derived from actual start and end times of transitions between indicated states based on a set of movies with varying lengths (movie durations in minutes: minimum, 85.2; maximum, 975; mean, 368; standard deviation, 210). (C) Probability distribution of zymosan state transitions color coded as above. The upper panel contains the distribution of transitions starting from surface bound zymosan (“S”). This probability was calculated over the entire course of time lapse observation. Of the zymosans that become fungipod associated, further transitions are possible as depicted in the lower panel showing state transitions starting from the fungipod associated zymosan (“F”) state. N = 301 total zymosan particles observed for a cumulative duration of ∼980 hours.
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Tables summarizing our findings regarding the mechanism of fungipod formation (A) and perspectives on the functional significance of fungipods (B). Abbreviations: SFK, Src-family kinases; PI3K, phosphoinositide 3-kinase; PTK, protein tyrosine kinase.
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Zymosan particle attachment and nascence of fungipods on immature DC. Red and green asterisks indicate a stably attached zymosan particle and points of fungipod generation, respectively. Timestamp refers to elapsed time of indicated zymosan particle's attachment. Playback speed is 250x faster than real time. Bar = 10 µm.
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Maturation of a fungipod formed by an immature DC with attached zymosan particles. Asterisk indicates a relevant zymosan particle. Green and blue arrowheads show nascent and mature fungipods, respectively. Timestamp refers to elapsed time of fungipod maturation. Playback speed is 250x faster than real time. Bar = 10 µm.
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Three-dimensional projection of actin stained within zymosan induced fungipods on DC showing the robust F-actin signal in the distal fungipod.
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DIC movie of an apparent rearward flow of refractile material in the distal fungipod. Playback at 10x faster than real time. Bar = 10 µm.
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DIC time-lapse showing supercoiling of a fungipod. Playback at 250x faster than real time. Bar = 5 µm.
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DIC time-lapse showing repeated driving of a fungipod-attached zymosan into the DC. The fungipod undergoes repeated cycles of kinking associated with displacements of the zymosan particle into the DC and relaxation of the fungipod. This zymosan is finally phagocytosed by the DC. Playback at 250x faster than real time. Bar = 10 µm.
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DIC movie of a chitin particle induced protrusion. Playback at 10x faster than real time. Bar = 10 µm.
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Three dimensional confocal projection of actin staining in a DC with zymosan particle (not visible) attached in a cup-like structure formed by numerous fungipods.
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Three-dimensional confocal projection of DiI staining in a DC with zymosan particle (not visible) enmeshed by fungipods.
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Three dimensional confocal projection of actin staining in a DC exhibiting several small zymosan-associated membrane wedges in addition to one large fungipod. Positions of zymosan particles are shown in the DIC insert image (insert bar = 10 µm).
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The authors thank Jim Bear, Richard Cheney, Keith Burridge, Con Beckers, Bill Goldman and Dick Anderson for insightful discussions; Pat Brennwald for live