BRIEF COMMUNICATIONS
Introduction
Methods
List of Markers
References
Authors
Building an Atlas of Subcellular Localization Markers in
WEHI-231 Cells
Chandy G, Mukai T, Mi Q, Zavzavadjian J, Gehrig E, Verghese M, Fung E, Couture
S, Park WS, O’Rourke N, Fraser I*
Alliance for Cellular Signaling Laboratories, Stanford University, Palo Alto, CA and California Institute
of Technology, Pasadena, CA
Abstract: A goal of the Alliance for Cellular Signaling (AfCS) Microscopy
Laboratory is to generate a visual library of subcellular structures that can
be used as a reference guide for localization studies. Our model system for
initial characterization of these markers is the WEHI-231 cell line. We have
collected a variety of fluorescently tagged protein markers that localize to
specific intracellular compartments, including cytosol, nucleus, nuclear
membrane, endoplasmic reticulum, Golgi, plasma membrane, and
cytoskeletal structures. This presentation briefly reviews our current
collection of subcellular markers and offers examples of their localization
pattern and temporal dynamics.
Published: January 22, 2003
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Introduction
An important approach to understanding the complexities of signal
transduction is studying the spatial organization of signaling molecules
among subcellular compartments. Knowledge of the localization patterns
and behaviors of these molecules can help define the regulatory
mechanisms utilized by a cell to control transmission of intracellular
signals. Our strategy for gathering information about subcellular
localization is, first, to express fluorescently tagged signaling molecules of
interest and make a subjective determination of their location using
confocal microscopy. Second, by choosing subsets of subcellular markers
that are also fluorescently tagged to coexpress with the signaling molecule,
we can then define specific localization patterns based on observed
colocalization. We will continue to expand our list of localization markers
as we encounter novel localization patterns. We present this brief overview
as an initial foundation for our atlas of subcellular markers.
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Methods
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Methods
Molecular biology: Specific molecules were chosen as markers primarily from
searching the literature for previously identified organelle markers. We used
localization domains instead of full-length proteins in cases where there is evidence that
those protein regions are sufficient to target a fluorescent tag to the location of interest.
When a full-length coding sequence was required, we used mouse sequences. Unless
otherwise specified, a single fluorescent protein was used as the fluorophore, and in this
report, only yellow fluorescent protein (YFP)-tagged markers were used. In two
instances, a 6-tandem YFP tag was used. Many of the markers described here are
available from Clontech. In the near future, we plan to make noncommercial marker
constructs available to the academic community and to provide further details of how
each was generated.
Cell culture and transfection: WEHI-231 cells were cultured in supplemented RPMI
medium 1640, as described in detail in the AfCS protocol PP00000111. Cells were
transfected with plasmid DNA by electroporation 16 to 24 hours prior to visualization
(PP00000113). For microscopy, cells were plated onto 8-well coverglass chambers
(PP00000114).
Imaging: Confocal images of cells were acquired with a cooled charge-coupled-device
(CCD) camera (Roper Scientific Photometrics CoolSNAP HQ) and viewed using a
Yokagawa spinning disk confocal head from PerkinElmer attached to the side port of a
Zeiss Axiovert 200 M microscope. Details of image acquisition and processing are
available in the AfCS protocol PP00000135.
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Methods
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Subcellular Markers
The following pages list and describe sample subcellular markers that are
currently used in the AfCS Microscopy Laboratory. Table 1 lists the
identity, specificity, and construct name for each marker. Images of
WEHI-231 cells expressing each of these markers follow. Images
represent three planes along the z-axis that were acquired to sample the
bottom, middle, and top of the cell fluorescence. Bright field images were
acquired at the same planes as the static fluorescent images. Time-lapse
images (30; 1 to 5 seconds apart) at one plane were acquired prior to the
static images. The range of fluorescent intensities that is represented on an
image is shown on the scale bar at the bottom left of each panel. The
intensity values vary along each plane and should be noted when
comparing images. Three examples of the utility of these markers are
shown on pages 18-20.
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Table 1. Sample markers for subcellular localization studies.
A05XM021A1NK
Centrosomes
γ-Tubulin
A08XM036B1TK
Plasma membrane
Farnesylation and polybasic motif of
K-Ras4b
A08XM005B1TK
Golgi
Golgin
A05XM011B1NK
Nuclear membrane
Lamin B receptor
A97XM003B2TK
Nucleus
YFP6-NLS
pEYFP-Nuc
Nucleus (with heavily
stained nucleolus)
NLS of SV40 large T antigen x 3
(Nuc)
A97GYFP6CXTK
Cytosol
Tandem fluorescent proteins (YFP6)
pEYFP-N1
Cytosol and nucleus
Single fluorescent protein
A08XM035A1TK
A08XM020A1TK
pEYFP-Mem
pEFYP-ER
Construct name
Microtubules
α-Tubulin
Polymerizing actin
Arp3
Plasma membrane and
perinuclear region
Myristylation and palmitylation
sequence of neuromodulin (Mem)
Endoplasmic reticulum
Calreticulin+KDEL
Subcellular region
Marker
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Cytosol and Nucleus
Fluorescent protein tags (alone) typically
distribute themselves throughout the
cytosol. This example shows that YFP
(Clontech, cat. no. 6006-1) is only excluded
from some vesicular and membranous
organelles that appear as a darker
“counterstain.” This marker is helpful for
the colocalization of signaling molecules
that distribute freely throughout the cell.
Help with movies
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Methods
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Cytosol
To create a marker that is excluded from
the nucleus, but is otherwise widely
distributed, we generated a construct with
six tandem repeats of YFP. This protein is
predicted to be close to 180 kD and is not
expected to passively diffuse into the
nucleus.
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Nucleus (with heavily stained nucleolus)
A construct with three tandem repeats of the
nuclear localization signal of simian virus
large T antigen joined to the C-terminus of
YFP produces a marker that specifically
targets the nucleus and demonstrates
enrichment in the nucleolus. The construct
encoding this chimera is available from
Clontech (cat. no. 6905-1).
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Nucleus (even distribution of fluorescent protein)
Three tandem repeats of the nuclear
localization signal of simian virus large T
antigen were joined to the C-terminus of six
tandem YFP molecules. This protein targets
to the nucleus but is not enriched in the
nucleolus.
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Introduction
Methods
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January 22, 2003
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Nuclear Membrane
The nuclear envelope is a dual membrane
contiguous with the the endoplasmic
reticulum. To identify the nuclear membrane,
a fluorescent protein was placed on the C-
terminal end of the first 238 amino acids of
the mouse lamin B receptor. The receptor
sequence consists of the nucleoplasmic tail
and the first transmembrane domain; thus,
the fluorescent tag is placed in the lumen of
the endoplasmic reticulum (1).
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Endoplasmic Reticulum
To identify the endoplasmic reticulum (ER),
we utilized a marker from Clontech (cat. no.
6906-1). This construct encodes the
fluorescent tag flanked by the ER targeting
sequence of calreticulin at the N-terminus
and the sequence for the ER retrieval
sequence, KDEL, at the C-terminus.
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January 22, 2003
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Golgi
This construct encodes a fusion protein
consisting of the C-terminal 72 amino acids
of Golgin-245 and the fluorescent protein.
This region contains a GRIP domain that
has been reported to bind the cytoplasmic
surface of Golgi membranes (2,3).
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Methods
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January 22, 2003
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Plasma Membrane (nonspecific)
To locate the plasma membrane, we used a
construct from Clontech (cat. no. 6917-1)
that encodes a fusion protein consisting of
the N-terminal 20 amino acids of
neuromodulin (GAP-43) and YFP. The 20-
amino-acid fragment contains a signal for
posttranslational palmitoylation of cysteines
3 and 4. This modification targets proteins to
membranes but not specifically to the plasma
membrane, as is evident by the intracellular
staining in these images.
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Introduction
Methods
List of Markers
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Plasma Membrane (specific)
A fusion protein of YFP and the C-terminal
20 amino acids of human K-Ras can achieve
increased plasma membrane specificity. The
20-amino-acid fragment contains a signal for
posttranslational farnesylation and a polybasic
motif (4). The diffuse cellular fluorescence
seen in the upper panel is from the plasma
membrane at the glass interface; thus, the
marker specificity is best appreciated in the
middle plane. The movie (below) was
captured near the glass surface to show the
membrane dynamics of this area.
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Introduction
Methods
List of Markers
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Microtubules
Microtubules are identified using a fusion
protein consisting of YFP and full-length
mouse α-tubulin. A similar construct
encoding human α-tubulin is available from
Clontech (cat. no.6118-1). Note that the
cytosol has similar intensity values in all
planes, but the much brighter tubular
structures seen in the middle panel increase
the image’s intensity range and make the
cytosol appear dimmer in that plane.
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Introduction
Methods
List of Markers
References
Authors
January 22, 2003
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Centrosomes
Centrosomes can be identified with a fusion
protein consisting of full-length mouse γ-
tubulin and YFP. In many cells the marker
appears to be purely cytosolic, but in some
cells one can clearly see one or two bright
punctate structures in one z position. Note
that the cytosol has similar intensity in all
planes, but the much brighter structures seen
in the middle panel increase the image’s
intensity range and make the cytosol appear
dimmer in that plane.
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Introduction
Methods
List of Markers
References
Authors
January 22, 2003
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Polymerizing Actin
This construct encodes a fusion protein of
YFP and the actin-related protein Arp3. The
Arp 2/3 complex concentrates at active sites
of actin polymerization (5).
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Methods
List of Markers
References
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January 22, 2003
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Utility of the Markers
Above left is a two-color overlay of the plasma membrane localization
sequence of K-ras, with an N-terminal cyan fluorescent protein (CFP)
tag, and G protein alpha 12 (Galpha 12; AfCS PID A000039), with an N-
terminal YFP tag. The marker colocalizes with the fraction of G protein
that is expressed in the plasma membrane, but there is a less prominent
cytosolic and nuclear fraction as well.
AfCS barcode for the YFP-G alpha 12 plasmid: A08XP025A1TK
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Methods
List of Markers
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January 22, 2003
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Utility of the Markers
Above left is a two-color overlay of lamin B receptor tagged with a C-
terminal CFP tag and a nemo-like serine-threonine kinase (NLK, AfCS
PID A001668) with a C-terminal YFP tag. In some cells, NLK is found
in a punctate pattern at the nuclear membrane. The lamin B receptor
identifies the nuclear membrane but does not fully “describe” the
localization pattern of NLK.
AfCS barcode for the NLK-YFP plasmid: A05XK050A1NK
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Methods
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Utility of the Markers
Above left is a two-color overlay of a cotransfection of γ-tubulin with a
C-terminal CFP tag and an Aurora 2 serine-threonine kinase (AfCS PID
A000352) with an N-terminal YFP tag. In some cells, the kinase is found
at one spot that localizes with γ-tubulin. This is consistent with previous
reports of Aurora-related kinases localizing to centrosomes (6).
AfCS barcode for the Aurora 2-YFP plasmid: A08XK104A1TK
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Introduction
Methods
List of Markers
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Table 2. Additional subcellular markers.
Other markers of the exocytic
and endocytic pathways
Vamp1a, 2, 4, 5, 7, 8, mammalian sec22,
mammalian YKT6
Recycling endosomes
VAMP 3/Cellubrevin
Intermediate filaments
Vimentin
Golgi
Galactosyltrasferase
Mitochondria
Cytochrome C-oxidase subunit 8a
ER-Golgi intermediate
compartment
ERGIC-53
Sorting endosomes
Tandem FYVE domain of HRS
Subcellular region
Marker
This table lists additional subcellular markers that have been or will be
tested in the laboratory, but for which images are not shown.
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References
1.
Ellenberg J, Siggia ED, Moreira JE, et al. (1997) J. Cell Biol. 138(6),
1193-1206. [PubMed]
2.
Kjer-Nielsen L, van Vliet C, Erlich R, Toh BH, and Gleeson PA.
(1999) J. Cell Sci. 112(Pt 11), 1645-1654. [PubMed]
3. Munro S and Nichols BJ. (1999) Curr. Biol. 9(7), 377-380. [PubMed]
4.
Prior IA and Hancock JF. (2001) J. Cell Sci. 114(Pt 9), 1603-1608.
[PubMed]
5. Weiner OD, Servant G, Welch MD, Mitchison TJ, Sedat JW, Bourne
HR. (1999) Nat. Cell Biol. 1(2), 75-81. [PubMed]
6.
Giet R and Prigent C. (1999) J. Cell Sci. 112(Pt 21), 3591-3601.
[PubMed]
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Methods
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* Please refer to the AfCS policy on authorship.
† To whom scientific correspondence should be addressed.
‡ AfCS Microscopy Lab, Stanford University, 975 California Avenue, Palo Alto, CA 94305
§ AfCS Molecular Biology Lab, California Institute of Technology, Pasadena, CA 91125
|| To whom questions or comments about the AfCS Communications should be addressed.
Henry Bourne
University of California San Francisco, San
Francisco, CA
Tobias Meyer
Stanford University, Palo Alto, CA
Reviewers
Ashley K. Butler
Duke University, Durham, NC
Gilberto R. Sambrano||
University of California San Francisco, San
Francisco, CA
Concept, Design, & Supervision
Grischa Chandy†‡
Iain Fraser† §
Nancy O’Rourke‡
Technical & Data Collection
Sam Couture §
Eileen Fung §
Elizabeth Gehrig‡
Qingli Mi §
Takako Mukai‡
Wei Sun Park‡
Mary Verghese‡
Joelle Zavzavadjian §
Editors
Authors
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Methods
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