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IMMUNOBIOLOGY
Lymphocyte microvilli are dynamic, actin-dependent structures that do not require
Wiskott-Aldrich syndrome protein (WASp) for their morphology
Sonja Majstoravich, Jinyi Zhang, Susan Nicholson-Dykstra, Stefan Linder, Wilhelm Friedrich, Katherine A. Siminovitch, and Henry N. Higgs
Short microvilli cover the surfaces of
circulating mammalian lymphocytes. The
surfaces of monocytes and neutrophils
are very different, containing ruffles as
their predominant structure. In this study,
we present the first quantitative characterization of lymphocyte microvilli. From
analysis of scanning electron micrographs, we find that median microvillar
length and surface density range from 0.3
to 0.4 ␮m and 2 to 4 microvilli/␮m2, respectively, on lymphocytes from a variety of
sources. As with similar structures from
other cells, lymphocyte microvilli contain
parallel bundles of actin filaments. Lymphocyte microvilli rapidly disassemble
when exposed to the actin-sequestering
molecule, Latrunculin A. This disassembly parallels cellular actin filament depolymerization and is complete within 2
minutes, suggesting that lymphocyte microvilli undergo continuous assembly and
disassembly. In contrast to previous reports suggesting lymphocyte microvillar
density to be reduced on lymphocytes
from Wiskott-Aldrich syndrome (WAS) patient, we find no such deficiency in either
mouse or human WAS protein (WASp)–
deficient lymphocytes. These results suggest that WASp is either not involved in or
is redundant in the rapid dynamics of
lymphocyte microvilli. (Blood. 2004;104:
1396-1403)
© 2004 by The American Society of Hematology
Introduction
Cell surfaces are rarely flat and generally contain a variety of
protrusions and/or invaginations. Microvilli are fingerlike protrusive structures found on the surfaces of many cells and are
abundant on the surfaces of circulating T and B lymphocytes.1
While definitive roles for lymphocyte microvilli have not been
established, a proposed function is in segregation of surface
receptors during extravasation.2 Two receptors involved in the
initial rolling phase of extravasation, L-selectin3,4 and ␣4␤7
integrin,5 localize to microvillar tips. In contrast, the ␤2 integrins
that mediate subsequent stable adhesion and diapedesis localize to
nonprotrusive regions of the cell surface.5 This spatial segregation
of surface receptors might enable a temporal segregation of
adhesive function during extravasation. Lymphocytes expressing
chimeric L-selectin constructs that no longer localize to microvilli
do not roll on L-selectin ligands,3 supporting this hypothesis.
Microvilli are correlated with metastatic potential of certain
cancers,6 perhaps due to their role in extravasation. In addition,
receptors for HIV, including CD4, CC chemokine receptor 5, and CXC
chemokine receptor 4, localize to microvilli,7 suggesting a potential role
for microvilli in HIV infection.
Other fingerlike protrusions on cell surfaces, including epithelial microvilli and surface-attached filopodia on many cell types,
depend on actin filaments for their structure.8,9 These filaments are
organized in parallel bundles that appear to span the length of the
microvillus and are cross-linked to each other and to the microvillar
membrane by a number of proteins.10 The dynamics of these
structures vary immensely. While filopodia grow and shrink
intermittently by alternate assembly and disassembly of their actin
filaments,11 epithelial microvilli maintain relatively constant length.
Nevertheless, the actin filaments in epithelial microvilli constantly
turn over the actin monomer subunits by a balance between
addition of new monomers at the distal tip and release of monomers
at the base.12
While lymphocyte microvilli also appear to be actin dependent,3
details of their structure and dynamics are unknown. Also unknown
are the mechanisms by which lymphocyte microvilli are assembled. Possible clues to this issue, however, are data suggesting
microvillar number to be reduced on lymphocytes from patients
with the Wiskott-Aldrich syndrome (WAS) immunodeficiency
disease.13-16 The defective protein in this disease is Wiskott-Aldrich
syndrome protein (WASp),17 a hematopoietic-lineage molecule and
an activator of the actin nucleation factor, Arp2/3 complex.18 These
findings suggest a pathway wherein WASp induction ofArp2/3 complex–
mediated actin filament assembly enables microvillar formation.
An as-yet-unanswered question in this model is how Arp2/3
complex, which causes formation of short, branched filaments,
could mediate formation of long, parallel actin filaments found in
microvilli. In migrating adherent melanoma cells, Arp2/3 complex
From the Department of Biochemistry, Dartmouth Medical School, Hanover, NH; the
Departments of Medicine, Immunology, and Medical Genetics and Microbiology,
Univerisity of Toronto, Toronto, ON, Canada; Samuel Lunenfeld and Toronto General
Research Institutes, Toronto, ON, Canada; Institut fuer Prophylaxe und
Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet,
Muenchen, Germany; and Universitätskinderklinik, Ulm, Germany.
Institutes of Health Research (CIHR) and a CIHR Senior Scientist Award
(K.A.S.); and grants from the Deutsche Forschungsgemeinschaft (SFB413,
GRK 438), Friedrich Baur Stiftung, and August Lenz Stiftung (S.L.). K.A.S. is a
Canadian Institutes of Health Senior Scientist.
Submitted February 4, 2004; accepted April 13, 2004. Prepublished online as
Blood First Edition Paper, May 6, 2004; DOI 10.1182/blood-2004-02-0437.
Reprints: Henry Higgs, Department of Biochemistry HB7200, Dartmouth Medical
School, Hanover, NH 03755-3844; e-mail: [email protected].
Supported by the Norris Cotton Cancer Center American Cancer Society
Institute grant IRG-82-003-18, by a Pew Biomedical Scholars grant, and by the
National Institutes of Health (NIH) grant P20RR16437 from the Centers of
Biomedical Research Excellence (COBRE) Program of the National Center for
Research Resources (H.N.H.). Also supported by a grant from the Canadian
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
1396
An Inside Blood analysis of this article appears in the front of this issue.
© 2004 by The American Society of Hematology
BLOOD, 1 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 5
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BLOOD, 1 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 5
appears to mediate filopodial formation indirectly through its role
in generating the leading edge lamellipodium.19 After Arp2/3
complex generates branched filaments, some of these filaments
are bundled and elongated, causing filopodia to protrude out of
the lamellipodium. However, blood lymphocytes are not adherent and contain no recognizable lamellipodia or ruffles, raising
doubts as to whether their microvilli could assemble through a
similar mechanism.
In this study, we examine lymphocyte microvilli in detail using
a quantitative scanning electron microscopy (SEM) assay. The data
reveal that lymphocytes from human blood and mouse blood, as
well as from a murine B-lymphoma culture line, possess similar
microvillar surfaces. These microvilli are highly dynamic, disassembling within 2 minutes when treated with the actin monomer
sequestering drug, Latrunculin A (LatA). The actin filaments in
lymphocyte microvilli are arranged in parallel bundles and are the
major actin-based structures in these cells. Lymphocytes from
several patients with severe WAS and from WASp-deficient mice
display microvillar characteristics similar to those of normal
lymphocytes. It therefore appears that development of these
structures on the lymphocyte cell surface can occur in the absence
of WASp activity.
LYMPHOCYTE MICROVILLAR PROPERTIES
1397
flash-frozen in liquid nitrogen and fixed in glutaraldehyde after thawing.
Mutations were as follows: patient 1, deletion of bases 107 and 108 of open
reading frame, resulting in a premature STOP at amino acid 36; patient 2,
121C⬎T, resulting in R41stop; patient 3, 291G⬎A, resulting in R86H; and
patient 4, undetermined. The presence of WASp in these lymphocytes was
not examined. All patients had clinically diagnosed severe WAS.
Human neutrophils (PMNs) were recovered from the pellets of Ficoll
gradients during lymphocyte isolation, washed 3 ⫻ in RPMI, suspended to
1 ⫻ 106/mL in medium, and incubated for 1 hour at 37°C/5% CO2. This
fraction contained 80% to 85% neutrophils by fluorescent antibody or light
scattering flow analysis (FACSCalibur), with less than 1% lymphocytes
or monocytes.
Human monocytes were isolated by counter-current elutriation.20 The
final cell population was suspended to 1 ⫻ 106/mL in medium and
incubated for 1 hour at 37°C/5% CO2.
Mouse lymphocytes were isolated from C57Bl/6 mice (normal lymphocytes) or from the WASp knock-out (KO) strain (WASp⫺/⫺ lymphocytes21).
Blood was drawn by cardiac puncture from mice killed by CO2 asphyxiation. A lymphocyte/monocyte population was isolated using LympholyteMammal (Cedarline Laboratories, Hornby, ON, Canada). Monocytes were
removed by plating on tissue culture plastic, as described for human
lymphocytes. The final cell population was suspended to 1 ⫻ 106/mL in
medium and incubated for 1 hour at 37°C/5% CO2.
The 300.19 cell line (Abelson-transformed murine pre-B lymphoma)
was obtained from Dr Geoffrey Kansas (Northwestern Medical School,
Chicago, IL) and cultured in RPMI1640 ⫹ 10% FBS ⫹ 0.05 mM
2-mercaptoethanol. For experiments, cells were used at 0.5 to 1 ⫻ 106/mL.
Materials and methods
Cells used
Human lymphocytes were isolated from healthy volunteers (Committee for
the Protection of Human Subjects [CPHS] 15406) under sterile conditions.
One unit of blood was drawn by venous puncture into acid citrate dextrose
(ACD), and mixed with Dextran T-500 (Amersham, Piscataway, NJ) to 2%
in a separatory funnel to aggregate erythrocytes. After 30 minutes, the lower
erythrocyte layer was drained and the upper layer was centrifuged for 10
minutes at 300g max in a swinging bucket rotor (same centrifugation
conditions used for subsequent steps unless indicated). The supernatant was
removed, and the pellet was resuspended in 30 mL iced lysis buffer (8.3 g/L
NH4Cl, 1 g/L NaHCO3, 0.04 g/L disodium EDTA [ethylenediaminetetraacetic acid]) and incubated on ice for 5 minutes. All subsequent steps were
carried out on ice or at 4°C. Cells were centrifuged and then washed 2 ⫻ in
300 mL 0.9% NaCl. The washed pellet was brought to 30 mL in 0.9% NaCl;
then 5 mL was layered carefully onto 7 mL Ficoll-Paque (Amersham) in six
15-mL polypropylene tubes (Fisher, Pittsburgh, PA). These step gradients
were centrifuged at 400g max for 15 minutes in a swinging bucket rotor.
The interfaces between NaCl solution and Ficoll were carefully removed,
pooled, diluted 5-fold in RPMI1640 ⫹ glutamine (Invitrogen, Frederick,
MD), and centrifuged. After 2 washes in RPMI, cells were resuspended to
1 ⫻ 106/mL in medium (RPMI ⫹ 10% fetal bovine serum [FBS; HyClone
defined; HyClone, Logan, UT]), plated onto 10-cm–diameter tissue culture
plates, and incubated for 1 hour at 37°C/5% CO2. Nonadherent cells were
centrifuged, resuspended in medium, and plated on tissue culture plastic a
second time. Nonadherent cells from the second plating were pelleted,
resuspended at 1 ⫻ 106/mL in prewarmed medium, and incubated at
37°C/5% CO2 for 1 hour before experimentation.
Preparations isolated in this manner contained 85% to 90% lymphocytes by forward- and side-scatter analysis in a FACSCalibur (Becton
Dickinson, San Jose, CA), the remainder being mostly smaller cells
(erythrocytes, platelets, and broken cells, 3%-5%), aggregates (1%-2%),
polymorphonuclear leukocytes (PMNs; 1.5%-2.5%), and monocytes (2%3%). Using fluorescently labeled antibodies and flow analysis (FACSCalibur), and gating out the smallest particles, cells were 88% to 93% T
lymphocytes (CD3⫹), 2% to 3% B lymphocytes (CD19), 2% to 3%
neutrophils (CD66b), and 0% to 1% monocytes (CD14).
Lymphocytes from severe human WAS patients and control subjects
were isolated by Ficoll-Hypaque gradient followed by plating onto tissue
culture plastic to remove adherent monocytes. Isolated lymphocytes were
Scanning electron microscopy
Cells were fixed in suspension from medium at 37°C/5% CO2. Cells (150
␮L) were added to 1.35 mL room temperature fixative (3.5% glutaraldehyde [EM Sciences, Fort Washington, PA] in 100 mM NaPO4 [pH 7.4]) and
rapidly mixed by inversion. After 2 hours of fixation, cells were centrifuged
in a swinging bucked rotor at 300g max for 5 minutes. The pellet was
washed twice in 1.5 mL NaPO4 (pH 6.0), then resuspended in less than 20
␮L of this buffer. This drop of cells was placed on top of a 5-mm round
acid-washed glass coverslip (EM Sciences) previously coated with 0.1%
poly-L-lysine (⬎ 300 000 molecular weight [MW]; Sigma, St Louis, MO).
After 30 minutes (while serenaded by a selection of baritone arias), cells on
the coverslip were treated with 0.5 mL fixative for 30 minutes. Coverslips
were postfixed in 0.5% osmium tetroxide for 1 hour, dehydrated in graded
ethanol washes followed by hexamethyldisilazane (HMDS; Aldrich),
desiccated overnight under vacuum, mounted onto 12-mm round aluminum
SEM stubs (EM Sciences), and gold-palladium sputter coated. Coverslips
were viewed on a Zeiss DSM 962 scanning electron microscope (Carl
Zeiss, Thornwood, NY) at 10 kV and 10-mm working distance.
Microvillar quantification from SEM images
Low-magnification images (⫻ 1000 or ⫻ 2000) were used to characterize
cell populations by morphology. Cells classified as possessing microvilli
had no apparent large or small ruffles. Microvilli were distinguished from
small ruffles by their appearance as fingerlike projections on cell sides.
“Damaged” cells were pockmarked or were missing large areas of plasma
membrane. Platelets, red blood cells, and reticulocytes were readily
discernable. High-magnification images (⫻ 10 000 or ⫻ 20 000) of individual cells were used for measuring microvillar lengths and densities,
using NIH Image software (NIH, Bethesda, MD). A 2 ⫻ 2 ␮m (1 ⫻ 1 ␮m
for mouse lymphocytes) box was created on top of the cell. The number of
microvilli that originated in the box was counted to determine microvillar
density. Individual microvilli within the box were traced to determine
microvillar length. Images were manipulated using Photoshop (Adobe,
San Jose, CA).
Light and fluorescence microscopy
Cells were fixed in suspension from medium at 37°C/5% CO2 in all cases.
Cells (150 ␮L) were added to 1.35 mL fixative (4% formaldehyde/0.05%
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1398
BLOOD, 1 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 5
MAJSTORAVICH et al
glutaraldehyde in phosphate-buffered saline [PBS]), mixed by inversion,
and fixed for 1 hour at 23°C. After washing twice in PBS, 100 ␮L cells was
spotted onto 12-mm round acid-washed coverslips coated with 0.1%
poly-L-lysine and incubated at 23°C for 30 minutes. Fixative (1 mL) was
added to coverslip and incubated for 15 minutes, followed by several PBS
washes. Cells were treated with 0.1 ␮M tetramethylrhodamine isothiocyanate (TRITC)–phalloidin (Sigma) in PBS with 1% calf serum and 0.1%
saponin for 1 hour at room temperature, then washed several times in PBS
and mounted in PVA/DABCO (Prolong Anti-Fade; Molecular Probes,
Eugene, OR). A Zeiss Axioplan2 microscope (Carl Zeiss) with ⫻ 100, 1.4
NA Plan-Apochromat objective was used. Hamamatsu (Bridgewater, NJ)
C4742-98 cooled CCD camera and OpenLab software (Improvision,
Lexington, MA) were used to collect a Z-series (0.5-␮m Z-sections).
OpenLab software was used to deconvolve the Z-series. Images were
manipulated using Photoshop (Adobe, San Jose, CA).
Transmission electron microscopy (TEM)
Cells were fixed in suspension from medium at 37°C/5% CO2. Cells (1 mL)
were added to 9 mL fixative (2.2% glutaraldehyde in 100 mM NaPO4 [pH
7.4]) and fixed for 2 hours. Cells were postfixed in 1% osmium tetroxide,
stained en-bloc with 0.5% uranyl acetate in water, dehydrated in a graded
ethanol series, embedded, and thin sectioned. Sections were stained with
2% uranyl acetate in methanol for 20 minutes, followed by lead citrate for 5
minutes, and then viewed on a JEOL 2000FX transmission electron
microscope (JEOL USA, Peabody, MA). Images were manipulated using
Photoshop (Adobe, San Jose, CA).
Cellular polymerized actin quantification
Cellular polymerized actin was quantified using TRITC-phalloidin following published methods.22 Briefly, a known number of cells was fixed in
suspension with 4% formaldehyde/0.05% glutaraldehyde in PBS for 1 hour,
washed in PBS several times, permeabilized with 1% TritonX-100 in PBS
for 5 minutes, stained with 1 ␮M TRITC-phalloidin in PBS with 1% bovine
serum albumin for 1 hour, washed in PBS several times, and extracted in 1
mL of 100% methanol overnight. The TRITC-phalloidin in the methanol
extract was quantified in an ISS PC1 spectrofluorometer (ISS, Champaign,
IL) at 546-nm excitation and 577-nm emission. A standard curve was
created from known concentrations of TRITC-phalloidin. Cytoplasmic
volume was calculated as described in the next paragraph.
Lymphocyte surface area and volume calculations
Surface area and cytoplasmic volume of human blood lymphocytes were
calculated by assuming radii of 2.7 and 1.5 ␮m, respectively, for the cell
and nucleus (measured from light, scanning, and transmission electron
micrographs). For 300.19 cells, these values are 4 and 2.5 ␮m. From these
values, the volume and surface area of human lymphocytes, assuming they
are spherical, are 6.83 ⫻ 10⫺14 L and 92 ␮m2, respectively. Our measured
microvillar density (4.1 per ␮m2; Table 2) means that human lymphocytes
possess on average 377 microvilli. Assuming a cylindric shape for
microvilli, with average radius of 50 nm and height of 380 nm (Table 2), the
volume and surface area of single microvilli are 2.98 ⫻ 10⫺18 L and 0.119
␮m2, respectively. Total microvillar volume and surface area are
1.12 ⫻ 10⫺15 L and 45 ␮m2, respectively. These values cause a 1.6%
increase in cytoplasmic volume, and a 49% increase in cell surface area.
Results
Microvilli are the predominant cell surface
feature on lymphocytes
We used scanning electron microscopy (SEM) to examine the
surface morphologies of human and mouse blood lymphocytes.
Our intent was to examine blood lymphocytes in suspension after
little prior manipulation to minimize cell surface alterations.
Viewed by SEM at low magnification, most cells from human
or mouse lymphocyte preparations (85% and 90%, respectively)
are spherical and display short microvilli (Figures 1A and 2A-B;
Table 1). A small proportion (11% for human and 7% for mouse) is
elongated and contains large membrane ruffles. A low percentage
of cells appears “damaged” (perforated or missing areas of plasma
membrane) and very low percentages of readily identifiable
erythrocytes or platelets are present. As observed previously,23
mouse lymphocytes (2.5-3 ␮m diameter) are significantly smaller
than are those from humans (5-6 ␮m).
In contrast to lymphocyte preparations, enriched fractions of
monocytes or PMNs have very different surface characteristics.
Most cells in monocyte preparations (84%) contain large ruffles as
their predominant surface feature (Figures 1B and 2E; Table 1).
These cells often display microvilli along with these large ruffles,
and sometimes microvilli are embedded in the ruffles. Most cells in
the PMN preparations display short ruffles throughout their surface
(Figures 1C and 2F; Table 1) and no readily identifiable microvilli.
About 20% of the cells in the PMN preparations are elongated and
highly ruffled at one end.
We used high-magnification SEM images (Figure 2) to quantify
both microvillar length and microvillar surface density on individual lymphocytes, selecting cells at random for this analysis.
Despite differences in cell diameter (about 5.5 ␮m for human
lymphocytes and 2.5 ␮m for mouse lymphocytes), both microvillar
parameters are similar between species (Table 2), with median
microvillar lengths of 0.3 to 0.4 ␮m and densities of 3 to 4
microvilli/␮m2. Neither population was sorted by lymphocyte
class, with the human pool being 90% T lymphocyte and 3% B
lymphocyte, and the mouse pool being 63% T lymphocyte and 32%
B lymphocyte (“Materials and methods”). Even so, the cell-to-cell
Figure 1. Low-magnification SEM of cells from human blood. (A) Lymphocytes. Arrow indicates cell with surface ruffles. (B) Monocytes. Cell 1 displays no ruffles but has
microvilli. Cell 2 displays ruffles on one half and microvilli on the other. (C) PMNs. Arrows indicate cells with large ruffles. Scale bars are 10 ␮m.
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BLOOD, 1 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 5
LYMPHOCYTE MICROVILLAR PROPERTIES
1399
Figure 2. Surface morphologies of white blood cells. SEMs of a human blood lymphocyte (A), mouse blood lymphocyte (B), 300.19 murine pre-B lymphoma cell (C), human
blood monocyte (E), and human blood PMN (F). Scale bars represent 2 ␮m. (D) Histogram of microvillar lengths for 300.19 cells (f), mouse blood lymphocytes (p), and human
blood lymphocytes (u). Bins are 0.05 ␮m. Numeric data are in Table 1.
variation in microvillar parameters is small, suggesting that T and
B cells have similar surface morphologies.
We also examined microvillar features on 300.19 murine pre-B
lymphoma cells, a line used extensively for studies of adhesion
receptors and cell rolling.4 These cells, although significantly larger
than either primary lymphocyte (8.2-␮m median diameter), possess
microvillar parameters similar to those of primary B and T cells
(Table 2). The length distributions of microvilli on lymphocytes
from these 3 cell types are also largely similar (Figure 2D).
Lymphocyte microvilli are dynamic, actin-dependent structures
Other fingerlike surface structures, such as epithelial microvilli and
growth cone filopodia, are dependent on long parallel bundles of
actin filaments,8,9 and past studies of lymphoma cell microvilli
suggest that they are actin dependent since they disassemble after a
30-minute treatment with high concentrations of cytochalasin D.3
We examined the actin dependence of lymphocyte microvilli in
more detail. Transmission electron microscopy demonstrates that
300.19 cell microvilli contain parallel assemblies of filaments of
similar diameter to actin filaments (Figure 3). Fluorescence microscopy of these cells, using the fluorescently labeled actin filamentbinding molecule, phalloidin, reveals intense staining at the cell
periphery and in microvillar protrusions (Figure 4A-B). Similar
staining is observed in human blood lymphocytes (not shown).
This staining suggests that microvilli are the major actin filamentcontaining structures in lymphocytes, since no ruffles or stress
fibers are readily visible.
The actin monomer-binding small molecule, Latrunculin A
(LatA), prevents actin monomer addition to filaments.24 Since
dynamic cellular actin filaments rapidly gain and lose monomers
from their ends,25 LatA causes depolymerization of dynamic
filaments by blocking polymerization, but affects stable filaments
much less rapidly. When added to 300.19 cells, LatA causes
cellular polymerized actin levels to drop by 75% within 1 minute
(Figure 4C). Surface microvilli disappear on a similar timescale
(Figure 4D). Upon LatA washout, microvilli recover on a slower
timescale (Figure 4E-G), possibly reflecting the high affinity of LatA for
monomers. These results demonstrate that microvilli are highly dynamic, growing and shrinking on a timescale of seconds to minutes.
Lymphocyte microvilli are not affected by lack of WASp
Previous studies using circulating lymphocytes or immortalized T
lymphocytes from human WAS patients suggested that WASp
deficiency resulted in decreased lymphocyte microvillar density
and altered morphology.13-15 Another study using splenic B cells
from WASp knock-out mice suggested a similar phenotype.16 We
analyzed circulating lymphocytes from WASp knock-out mice
using our quantitative SEM assay. Surprisingly, we found no
Table 1. Low-magnification (ⴛ 2000) SEM characterization of white blood cells
Cell type
No. of
cells
% villi
% small
ruffles
% large
ruffles
% smooth
% RBCs
Human lymphs
1382
85
0
11
3
0
2
Human PMNs
774
0
77
20
2.5
0
1
2
% platelets
Human monos
120
13
0
84*
3
0
Mouse lymphs
174
90
2
7
0
1
0
300.19 cells
325
84
0
0
16
—
—
RBCs indicates red blood cells; PMN, polymorphonuclear leukocytes (neutrophils); monos, monocytes; and —, not applicable.
*Of these cells, 62% had at least one obvious microvillus.
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MAJSTORAVICH et al
Table 2. Microvillar parameters of lymphocytes
Cells
Median microvillar
length, ␮m
Length standard
deviation (n)
Microvillar density,
villi/␮m2
Density standard
deviation (n)
300.19 cells
0.39
0.27 (1267)
2.9
1.3 (27)
Human lymphs
0.38
0.22 (1200)
4.1
1.2 (28)
Mouse lymphs
0.31
0.14 (310)
4.1
1.0 (19)
Mouse WASp KO
0.31
0.16 (227)
3.3
1.6 (17)
Human healthy control
0.30
0.11 (212)
4.4
2.1 (6)
Human WAS 1
0.29
0.13 (404)
6.4
1.0 (6)
Human WAS 2
0.30
0.15 (277)
4.8
0.7 (7)
Human WAS 3
0.31
0.10 (277)
5.6
1.4 (7)
Human WAS 4
0.30
0.10 (396)
4.0
1.5 (11)
Human healthy control indicates human lymphocytes prepared in the same manner as WAS lymphocytes; human WAS 1 to 4, lymphocytes from WAS patients.
difference in microvillar length and only a slight (20%) decrease in
microvillar density (Figure 5; Table 2).
We also obtained lymphocyte preparations from 4 different
human patients clinically diagnosed as displaying severe WAS. The
genotypes of 3 patients had been determined. There are 2 patients
who are predicted to express no WASp, since they possess
mutations resulting in premature stop codons at amino acids 36 and
41, respectively. The third patient contains a missense mutation
resulting in R86H, an acknowledged mutational hot spot that has
been identified as causing severe WAS in other patients.17 Lymphocyte microvillar lengths and densities from all patients were
indistinguishable from those of healthy subjects isolated by the
same procedure (Figure 6; Table 2).
Discussion
In this study, we present the first quantitative characterization of
lymphocyte microvilli. These short (300-400 nm) structures are
abundant (3-4 per ␮m2) on the surfaces of lymphocytes from
several sources. From our measurements, microvilli increase the
surface area of human blood lymphocytes appreciably (49%),
while having little effect on cytosolic volume (1.6% increase;
“Materials and methods”). In addition to preferential localization of
rolling receptors to microvilli,3-5 this increase in surface area might
enhance the ability of these cells to roll.
Previous publications using TEM suggested that both monocytes and PMNs possessed surface microvilli.26-28 Other work
using SEM revealed that PMNs expressed ruffles predominantly on
their surface29,30 and pointed out that distinguishing between
microvilli and ruffles by TEM was difficult. Due to the small sizes
of the protrusions on PMNs and lymphocytes, we are not able to
declare definitively that PMNs possess ruffles exclusively or that
lymphocytes do not possess any ruffles. However, our work
suggests that the dominant surface protrusions on lymphocytes are
microvilli, while those on PMNs are predominantly ruffles. The
protrusions on monocytes are much larger and are predominately
ruffles with some microvilli interspersed.
While both microvilli and ruffles are actin dependent, these
structures differ greatly in their actin filament architecture and
the proteins that mediate these architectures. Ruffles contain a
meshwork of short filaments cross-linked in an end-to-side
manner, these cross-links being mediated by Arp2/3 complex
and filamin.31 In contrast, the long, parallel filament bundles in
microvilli are cross-linked side-to-side by proteins such as
fimbrin, villin, fascin, and espins.10 These architectural differences strongly suggest major differences in regulation of
assembly/disassembly of these structures, so to consider them
equivalent would be erroneous. However, L-selectin localizes
both to microvillar tips on lymphocytes and to ridges of ruffles
on PMNs,3,4,29 so the 2 structures might be functionally equivalent in this respect.
The small size of lymphocyte surface protrusions makes it
difficult to test whether lymphocytes contain some small ruffles in
addition to microvilli or if PMNs contain microvilli embedded in
their small ruffles. Resolution of this question will be critical to
determining assembly mechanisms for these structures. Recent
studies on filopodia in melanoma cells, which contain parallel
filaments like microvilli, suggest that filopodial filaments might be
generated from within a substantial rufflelike actin filament network. Short filaments generated by Arp2/3 complex in this network
are bundled by the cross-linking protein, fascin, and elongate to
Figure 3. Lymphoma cell microvilli contain parallel actin filaments.
Transmission electron micrographs of a 300.19 cell (A) and of an individual
300.19 cell microvillus (B). Note parallel striations in microvillus, which
have diameters similar to those of actin filaments. Scale bars represent 1
␮m (A) and 50 nm (B).
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BLOOD, 1 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 5
Figure 4. Lymphoma cell microvilli are dynamic and actin dependent. (A)
Phase-contrast micrograph of a fixed 300.19 cell. (B) Rhodamine fluorescence of
same 300.19 cell stained with rhodamine-phalloidin. (C) Time course of decrease in
cellular rhodamine-phalloidin after treating 300.19 cells with 1 mm LatA or with
dimethyl sulfoxide (DMSO) carrier. (D) SEM of 300.19 cell 2 minutes after treatment
with 1 ␮M LatA. (E-G) SEMs of 300.19 cells before treatment (E), after 10 minutes in
1 ␮M LatA (F), and 30 minutes after LatA wash out (G). Scale bars are 2 ␮m. Original
magnification ⫻ 1000 (A-B) and ⫻ 10 000 (D).
form filopodia.19 From our SEM and TEM images, no comparable
extensive actin filament network exists in lymphocytes, but we
cannot rule out the possibility that a more modest network serves
the same purpose.
The rapid retraction of microvilli upon LatA treatment suggests
that these structures are highly dynamic, growing and shrinking on
a timescale of seconds to minutes. LatA sequesters actin monomers
but does not actively depolymerize filaments. Thus, lymphocyte
microvilli must constantly cycle between assembly and disassem-
Figure 5. Lymphocytes from WASp knock-out mice
have normal microvilli. (A) SEM of representative blood
lymphocyte from WASp knock-out mouse. Scale bar
represents 2 ␮m. (B) Histogram comparing microvillar
lengths for wild-type and WASp knock-out mouse lymphocytes. Bins are 0.05 ␮m. Numeric data are in Table 2.
LYMPHOCYTE MICROVILLAR PROPERTIES
1401
bly, driven by polymerization and depolymerization of actin
filaments. When LatA blocks polymerization, filaments depolymerize due to normal depolymerization mechanisms. Their dynamics
suggest that lymphocyte microvilli are able to adapt rapidly to
signals. Such structures might be more widespread than is generally appreciated, as several cell types appear to possess short,
dynamic microvilli.32
Our results suggest that WASp deficiency in either mouse or
human lymphocytes does not result in dramatic microvillar length
or density defects in circulating lymphocytes. These findings differ
from data published previously in several reports,13-16 which may
reflect differences in cell preparative techniques in the different
studies. One study used SEM to examine allo-specific T lymphocytes isolated from WAS patient blood and cultured for several
months in the presence of interleukin-2 (IL-2)– and mitomycin
C–treated Raji cells.14 As major changes to T-cell cytoskeleton
occur upon T-cell activation, and WASp plays an important role in
this process,21,33 this latter finding may reflect features of activated
rather than unstimulated T cells. Similarly, published data on
microvillar structure on splenic B cells from WASp KO mice
involved analysis of cells treated with lipopolysaccharide or with
anti-CD40 and IL-4.16 One other report on lymphocyte microvillar
structures from WAS patients did involve analysis of peripheral
lymphocytes isolated and prepared in a similar manner to those in
our work.13 However, the WAS gene had not been identified at that
time and molecular characterization of the patients was not possible.
Moreover, the microvillar defects observed in that study were mild, and
the data may not substantively differ from our results.
In summary, WASp does not appear crucial for maintaining
microvillar morphology on lymphocytes. One possible explanation
for these findings is that WASp effects on microvillar actin
filaments can be achieved through other effector pathways as well.
The recently identified actin assembly abilities of formin proteins34
suggest some redundancies between formins and WASp functions
and raise the possibility that formins compensate for WASp in
microvillar regulation. Similarly, N-WASP activity may also compensate for the absence of WASp in relation to modulating
microvillar dynamics. Alternately, WASp may be more relevant in
activated cells, just as WASp effects on immunologic synapse
formation arise only in the context of T-cell receptor engagement.21,33 Resolving these issues will require further investigation
into the effectors that localize to microvillar structures and
definition of the extent to which WASp effects on actin remodeling
overlap with effects of other cytoskeletal modulatory proteins.
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1402
BLOOD, 1 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 5
MAJSTORAVICH et al
Figure 6. Lymphocytes from human severe WAS patients
have normal microvilli. (A,C) SEMs of representative blood
lymphocytes from 2 patients with severe WAS. Scale bars
represent 2 ␮m. (B,D) Histograms comparing microvillar lengths
from normal human lymphocytes and those from the respective
WAS patient. Bins are 0.05 ␮m. Numeric data are in Table 2.
Acknowledgments
We are greatly indebted to the WAS patients and their families for
contributing to these studies. We also thank Peter Morganelli (VA
Hospital, White River Junction, VT) and Paul Wallace (formerly at
Dartmouth Medical School, now at Roswell Park Cancer Center) for
contributing purified monocytes; Dr Charles Daghlian and Louisa
Howard in the Ripple Electron Microscope Facility at Dartmouth for
their expertise and advice; Duane Compton and his laboratory for use of
his fluorescence microscope and lots of advice; Peter C. Weber for
continuous support; and Barbara Boehlig for technical support.
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2004 104: 1396-1403
doi:10.1182/blood-2004-02-0437 originally published online
May 6, 2004
Lymphocyte microvilli are dynamic, actin-dependent structures that do
not require Wiskott-Aldrich syndrome protein (WASp) for their
morphology
Sonja Majstoravich, Jinyi Zhang, Susan Nicholson-Dykstra, Stefan Linder, Wilhelm Friedrich,
Katherine A. Siminovitch and Henry N. Higgs
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