Dual channel STED nanoscopy of lytic granules on actin filaments in natural killer cells.

2012·0.98 MB·English

by  Mace, Emily M.

Communicative&IntegrativeBiology5:2,184–186;March/April2012;G2012LandesBioscience
Dual channel STED nanoscopy of lytic granules on actin filaments
in natural killer cells
Emily M. Mace1 and Jordan S. Orange1,2,*
1Children’sHospitalofPhiladelphiaResearchInstitute,Philadelphia,PAUSA;2UniversityofPennsylvaniaSchoolofMedicine,Philadelphia,PAUSA
Natural killer (NK) cells are innate target, polarization of the MTOC and
immune effectors that eliminate directed secretion of lytic granules. Char-
diseased and tumorigenic targets through acterization of the immunological synapse
thedirectedsecretionofspecializedsecre- by 3D reconstruction of confocal images
tory lysosomes, termed lytic granules. suggested a dense ring of peripheral actin
This directed secretion is triggered with a paucity of central actin, allowing
following the formation of an immuno- for secretion of granules through the void
logical synapse (IS), which is character- in the center.2 However, it has previously
ized by actin re-modeling and receptor been shown that the actin-associated
organization at the interface between the motor protein myosin IIA is required for
© 2012 Landes Bioscience.
NKcellanditssusceptibletarget.Actinat degranulation in NK cell cytotoxicity.3
theIShasbeendescribedtobepermissive In subsequent studies, we determined
to secretion by forming a large central that myosin IIA is directly associated with
Do not distribute.
clearance through which lytic granules NK cell lytic granules and is required for
are released. However we, and others, their ability to interact with actin fila-
have recently shown that the actin net- ments.4 This suggested that granules are
work in NK cells at the IS is dynamic associated with actin prior to delivery to
yet pervasive. These efforts used multiple the plasma membrane. We hypothesized
high resolution imaging techniques to therefore that F-actin would be present
demonstrate that the actin network does in central regions of the IS and would
not act as a barrier to secretion, but serve a valuable function in directly
instead enables the secretion of lytic interactingwithlyticgranules.Inpursuing
granules through minimally sized clear- this question, we recently demonstrated
ances. In our recent publication we that F-actin is, indeed present in the
visualized actin using continuous wave central region of the IS, but had
stimulated emission depletion (CW- been previously undetected due to the
STED) and lytic granules using the limitations of conventional fluorescence
Keywords: natural killer cells,
confocal modality. Here we report for microscopy.5,6
STED microscopy, lytic granules, actin,
the first time dual channel STED nano- While the diffraction barrier of light
immunological synapse
scopy of NK cell lytic granules on actin has previously limited the resolution of
Submitted: 11/18/11 filaments. microscopy, new advances in imaging
Accepted: 11/21/11 have resulted in an explosion of technolo-
gies enabling the spatial resolution of
http://dx.doi.org/10.4161/cib.18818
As potent effector cells of the innate structures less than 200 nm.7 One such
*Correspondenceto:JordanS.Orange;
immune system that rely on germline technology is STED, which employs a
Email:[email protected]
encoded receptors for activation, NK cells toroidal-shaped depletion laser beam that
must pass tightly regulated checkpoints to temporarily depletes fluorescent emission
Addendumto:RakGD,MaceEM,BanerjeePP,
the formation of a mature immunological around the fluorophore, thus enabling
SvitkinaT,OrangeJS.Naturalkillercelllytic
granulesecretionoccursthroughapervasive synapse and subsequent cytotoxicity.1 resolutionofobjectsseparatedbylessthan
actinnetworkattheimmunesynapse.PLoSBiol These checkpoints include the rearrange- 50nm.8Inourrecentwork,weemployed
2011;9:e1001151;PMID:21931536;http://dx.doi. ment of filamentous (F-) actin at the multiple high-resolution imaging techni-
org/10.1371/journal.pbio.1001151
interface between the NK cell and its ques, including total internal reflection
184 Communicative&IntegrativeBiology Volume5Issue2
ARTICLEADDENDUM
fluorescence microscopy, platinum replica greater resolution in STED, with a The increased resolution we were able
electron microscopy and CW-STED to FWHM value of 90 nm, whereas to identify using STED resulted in an
demonstrate comprehensively that F-actin FWHM in confocal was 210 nm. ability to define lytic granules of an
ispresent throughouttheIS.5Inaddition,
we reported confocal microscopy of lytic
granules on actin filaments detected by
STED. We have since optimized dual
color STED detection and here report the
imagingofbothNKcelllyticgranulesand
F-actin in STED.
Imaging of Lytic Granules
on Actin Filaments in Confocal
and Dual Color STED
In order to recapitulate the lytic IS in an
alignment suitable for super-resolution
imaging, we utilized glass coated with
antibodies directed against the NK cell
activating receptor NKp30 and adhesion
receptor CD18, as described previously.5
The human NK cell line, NK92, was
prepared in single cell suspension and
adhered to antibody-coated glass for
© 2012 Landes Bioscience.
20 min then fixed. After fixation, cells
werepermeabilizedandstainedforF-actin
using phalloidin Alexa Fluor 488 and for
Do not distribute.
thelyticgranulecomponentperforinusing
Pacific Orange-conjugated anti-perforin
antibody. Using sequential scanning, we
evaluated actin via phalloidin Alexa
Fluor 488 in STED and anti-perforin
via the Pacific Orange secondary antibody
in both STED and confocal imaging
modes. Images were acquired using
Leica ASAF software then exported to
Volocity software (Perkin Elmer) and
thresholded using the same settings in all
cases to allow for quantitative comparison
of the images.
As we had previously identified, both
F-actin and lytic granules were present
throughout the synapse.5 There was a
qualitative improvement in the resolution
of the lytic granules imaged using the
STED modality (Fig.1A, red), when
compared with confocal (Fig.1B, red).
To quantitatively compare resolution, we Figure1.VisualizationoflyticgranulesimagedbyCW-STEDandconfocalonF-actin.NK92cells
measured a single granule in both STED wereadheredtoglasscoatedwithantibodytoactivating(NKp30)andadhesion(CD18)receptor
thenfixed,permeabilizedandstainedforperforinandactin.CellswereimagedusingCW-STED
and confocal and determined the full
(actin,green)andeitherCW-STEDorconfocal(perforin,red).Shownisthesamecellwithgranules
width at half maximum (FWHM) using
detectedbySTED(A)orconfocal(B).Aregionofinterestisenlargedtoshowgreaterresolutionof
Leica ASAF software (Fig.1C). FWHM granules(centerpanel).(C)Fullwidthhalfmaximum(FWHM)measurementsofconfocal(green
measures the width of the fluorescence line)andSTEDimages(redline).Horizontaldashedlinesshowhalfmaxima,verticaldashedlines
intensity peak and thus reflects the ability showwidthathalfmaxima.(D)Representativelineprofileofpixelintensitiesofactin(greenline)
andperforin(redline)takenfromalinebisectingasinglegranule(showninwhiteinSTEDimage
toseparateorresolveobjects.Assuggested
enlargement).AU,arbitraryunits.
by our observations, analysis confirmed
www.landesbioscience.com Communicative&IntegrativeBiology 185
apparent smaller size. Thus, with this cells.9-12 It is required for cell surface if this model extends to other immune
improved ability to distinguish lytic receptor rearrangements, cell activation cells undergoing directed secretion of
granules, we sought to confirm our earlier signaling and for the subsequent polariza- both specialized secretory lysosomes and
findingthatlyticgranules,whilelocatedin tion of lytic granules to the IS. Previous cytokines. Alternatively, it may represent
areas of F-actin hypodensity, were either studiesperformedusing3Dreconstruction an additional checkpoint utilized by cells
in minimally sized clearances in contact ofconfocalimages,however,haveresulted of the innate immune system as they
with or atop F-actin filaments. In order in a model for secretion in which lytic access pre-armed functions. Using dual
to accomplish this, we measured line granules are expelled through a central color STED nanoscopy of lytic granules
profiles of fluorescence intensity for per- clearance of actin in both NK cells and on actin filaments in NK cells we have
forin and F-actin staining. Consistent their adaptive counterpart, the cytotoxic shown in unprecedented resolution details
with our earlier findings, we found an T lymphocyte.2,3,13,14 Detailed super- of this new paradigm.
intersectionoflineprofiles(Fig.1D).This resolution analysis of the NK cell lytic
indicates that lytic granules are closely synapse by two independent laboratories Acknowledgments
associated with F-actin and thus secreted suggests a new paradigm for cytotoxicity This work was supported by NIH R01
through minimally sized clearances. in which a pervasive actin network is AI67946(awardedtoJ.S.O.).Theauthors
Actin reorganization at the IS is a present and acts not as a barrier but a wish to thank Geoff Daniels at Leica
critical prerequisite for cytotoxicity in facilitator for secretion.5,6 With this new Microsystems for technical assistance and
both adaptive and innate immune effector understanding it will be interesting to see Dr. G. Rak for helpful discussion.
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