RESEARCH ARTICLE

Cytoskeleton, May 2016 73:258–268 (doi: 10.1002/cm.21299)

C
V 2016 Wiley Periodicals, Inc.

Myo1g Is An Active Player in Maintaining

Cell Stiffness in B-Lymphocytes
opez-Ortega,1,2 E. Ovalle-Garcıa,3 I. Ortega-Blake,4 A. Antill
O. L on,4 B. Chavez-Munguıa,5
G. Pati~ opez,6 R. Fragoso-Soriano,7 and L. Santos-Argumedo1*
no-L
1 n Y De Estudios Avanzados Del Instituto Politecnico Nacional,
Departamento De Biomedicina Molecular, Centro De Investigacio
Ciudad De Mexico, C. P. 07360, Mexico
2 noma De Mexico, Ciudad De Mexico, C. P. 04510, Mexico
Facultad De Medicina, Universidad Nacional Auto
3 noma De Nuevo Leo n, UANL. Facultad De Ingenierıa Mecanica Y Electrica, Av. Universidad S/N, Ciudad Universitaria,
Universidad Auto
San Nicolas De Los Garza, Nuevo Leo n, C. P. 66451, Mexico
4
Instituto De Ciencias Fısicas, UNAM, Cuernavaca, Morelos, C. P. 62210, Mexico
5 mica Y Patogenesis Molecular, Centro De Investigacion Y De Estudios Avanzados Del Instituto Politecnico
Departamento De Infecto
Nacional, Ciudad De Mexico, C. P. 07360, Mexico
6 n En Inmunologıa Y Proteo
mica, Hospital Infantil De Mexico, “Federico Gomez”, Ciudad De Mexico, C. P.
Laboratorio De Investigacio
06720, Mexico
7 n Y De Estudios Avanzados Del Instituto Politecnico Nacional, Ciudad De Mexico, C. P.
Departamento De Fısica, Centro De Investigacio
07360, Mexico
Received 26 October 2015; Revised 11 April 2016; Accepted 13 April 2016
Monitoring Editor: Manuel Thery

B-lymphocytes are migrating cells that specialize in anti- Introduction

gen presentation, antibody secretion, and endocytosis;
these processes implicate the modulation of plasma
membrane elasticity. Cell stiffness is a force generated by
the interaction between the actin-cytoskeleton and the
plasma membrane, which requires the participation of
several proteins. These proteins include class I myosins,
which are now considered to play a role in controlling
membrane–cytoskeleton interactions. In this study, we
identified the motor protein Myosin 1g (Myo1g) as a
mediator of this phenomenon. The absence of Myo1g
decreased the cell stiffness, affecting cell adhesion,
cell spreading, phagocytosis, and endocytosis in B-
lymphocytes. The results described here reveal a novel
molecular mechanism by which Myo1g mediates
and regulates cell stiffness in B-lymphocytes. V 2016 Wiley
C et al., 2012].
Periodicals, Inc.
Key Words: B-lymphocyte; cell stiffness; cytoskeleton;
myosin
Additional Supporting Information may be found in the online ver-
sion of this article.
*Address correspondence to: Dr. Leopoldo Santos-Argumedo, Depar-
tamento de Biomedicina Molecular, Centro de Investigacion y de
Estudios Avanzados del IPN, Avenida IPN 2508, C.P. 07360, Ciu-
dad de Mexico, Mexico. E-mail: lesantos@cinvestav.mx
Published online 22 April 2016 in Wiley Online Library
(wileyonlinelibrary.com).
I mmune surveillance involves the mobilization of cells
through different organs and tissues, where the cells are
exposed to different mechanical pressures [Olety et al.,
2010]. The cells respond to external stimuli by modulating
their elasticity. Cell stiffness, is regulated by the interactions
between the actin-cytoskeleton and the plasma membrane
[Olety et al., 2010]. This interaction is important during
cell spreading [Raucher and Sheetz, 2000], cytokinesis,
phagocytosis, endocytosis, cell migration [Raucher and
Sheetz, 2000], cell adhesion [Keren, 2011], vesicular traf-
ficking [Keren, 2011], and the development of membrane-
protrusions, such as microvilli [Pietuch et al., 2013], filopo-
dia [Heiman and Shaham, 2010], and pseudopods [Houk
The lipid membrane is non-elastic and cannot sustain
large pressures [Staykova et al., 2011]. Therefore, cells
respond by adding or removing plasma membrane through
exocytosis or endocytosis, thereby significantly modifying
cell rigidity [Staykova et al., 2011]. B-lymphocytes are spe-
cialized cells that are involved in antibody secretion, and
the endocytosis and exocytosis of several molecules; all these
phenomena lead to an important exchange of membranes
between the plasma membrane and intracellular compart-
ments. Various proteins, membrane lipids, and cell firmness
regulate this mobilization of membranes.
To achieve the mobilization of membranes, the presence
of proteins that bind the actin cytoskeleton to the plasma
membrane is important. Myosins are known to play an

䊏 258
important role in this phenomenon [Olety et al., 2010].
They are the primary microfilament–associated proteins
and comprise a family of molecules characterized by both
their ability to bind to filamentous actin and their ATPase
activity, which couples the hydrolysis of ATP with confor-
mational changes. These changes direct the movement of
myosins along the microfilament cytoskeleton; simultane-
ously, the myosins can also specifically bind various mem-
brane phospholipids. Consequently, myosins, such as class I
myosins, represent probable candidates for mediating the
maintenance of cell stiffness [Krendel, 2005].
The class I myosin family has eight members (Myo1a–
Myo1h) [Kim et al., 2006; Maravillas-Montero and Santos-
Argumedo, 2012]. Previous work has shown that the
knockdown of class I myosins in Dictyostelium discoideum
decreases cortical tension whereas the overexpression of
these proteins has an opposite effect [Dai et al., 1999].
Enterocytes of Myo1a-deficient mice have lower cell mem-
brane tension than that of the wild-type (WT) cells, and
the reconstitution of class I myosin in these cells results in
an increase in this parameter [Nambiar et al., 2009].
Myosin1g (Myo1g) is a monomeric class I myosin that
comprises: a single N-terminal catalytic motor (head)
domain; a regulatory neck region containing 2 IQ-motifs
for calmodulin binding; and a C-terminal tail that directly
associates, through a pleckstrin homology (PH domain),
with phosphatidylinositol 3,4-bisphosphate [Patino-Lopez
et al., 2010] and phosphatidylinositol 3,4,5-triphosphate
[Dart et al., 2012] in membranes.
Myo1g is expressed in hematopoietic cells, especially in
B-lymphocytes [Patino-Lopez et al., 2010; Maravillas-
Montero and Santos-Argumedo, 2012; Maravillas-Montero
et al., 2014], and localizes to the plasma membrane [Pat-
ino-Lopez et al., 2010]. It is required for the maintenance
of membrane tension in T-lymphocytes [Olety et al., 2010;
Gerard et al., 2014], and is also involved in the phagocyto-
sis of opsonized microbeads in macrophages [Dart et al.,
2012]. In a previous paper, we reported that Myo1g is
involved in spreading, adhesion, endocytosis, and exocytosis
[Maravillas-Montero et al., 2014]. In that paper, we
described defects in cell functions that involve the regula-
tion of cell stiffness. Therefore, it is probable that these
abnormalities in maintaining cell elasticity or membrane
tension may be due to the lack of Myo1g, because the par-
ticipation of class I myosins in this phenomenon is well
known [Olety et al., 2010; Gerard et al., 2014]. This
hypothesis arises from the importance of class I myosins in
maintaining membrane tension in several cell types, such as
fibroblasts [Houk et al., 2012], where the authors showed
defects in cell spreading due to changes in membrane ten-
sion. Similarly, in endothelial cells, changes in membrane
tension affect cell adhesion [Delanoe-Ayari et al., 2004].
Finally, it has been reported that modifications in the mem-
brane tension of MDCK [Boulant et al., 2011] and HeLa
CYTOSKELETON
cells [Raucher and Sheetz, 1999] affects their endocytosis
capability.
We have illustrated several defects in B-cells obtained
from Myo1g-deficient mice; however, the mechanism by
which Myo1g regulates these phenomena is unclear [Mara-
villas-Montero et al., 2014]. For this reason, our goal in
this work was to evaluate cell stiffness and cell functions in
Myo1g2/2 and WT B-cells where membrane elasticity is
important. Thus, the aim of this study was to characterize
the function of Myo1g in maintaining cell stiffness in B-
lymphocytes. Depletion of Myo1g leads to a loss of cell
stiffness, affecting endocytosis, cell spreading, and cell adhe-
sion. These results reveal a novel molecular mechanism by
which Myo1g mediates and controls cell stiffness in B-cells,
which is important for several processes.
Results
Lack of Myo1g Alters Cell Adhesion
and Spreading in B-Lymphocytes
To evaluate the role of Myo1g in the maintenance of cell
stiffness in B-lymphocytes, we quantified this parameter in
the B-lymphocytes of WT and Myo1g-deficient mice using
an atomic force microscope (AFM). This methodology is
widely used for evaluating biophysical parameters in several
cell types, allowing the quantification of resistance to cell
deformation in live cells. The resistance to cell deformation
illustrates the link between the actin-cytoskeleton and the
plasma membrane, and this opposition is an important fea-
ture of cell physiology. This methodology is graphically
described in Fig. 1a. We evaluated cell stiffness in resting
and activated B-lymphocytes. We obtained 16 force-
nanoindentation curves (Fig. 1b), we processed these curves
to obtain the real values of cell stiffness. As shown in Fig. 1c,
cell stiffness was higher in the WT B-cells than in the
Myo1g2/2 B-cells when both cell types were activated. Inter-
estingly, under resting conditions, the WT and Myo1g2/2
B-cells showed the similar values.
We analyzed CD44- and IgM-dependent adhesion and
spreading. In the spreading assays, we consistently observed a
clear defect in the CD44-dependent spreading of Myo1g2/2
B-cells; this defect was not observed in the IgM-induced
spreading (Fig. 1d). A more detailed view of this process can
be observed in the enlarged pictures (white arrows).
To quantify these differences, we evaluated the spread
area of each lymphocyte from a total of 200 cells per group.
As depicted in Fig. 1e, the Myo1g2/2 B-cells did not
spread as much as the WT cells when an a-CD44 stimulus
was used; however, we did not see these differences with an
a-IgM stimulus. To verify these results, we also quantified
the “shape factor”, which was the coefficient of the length
and width of each given cell. Values closer to 1.0 indicated
a more rounded shape compared with the WT cells. As
shown in Fig. 1f, most of the Myo1g2/2 B-cells developed
Myo1g Mediates Cell Stiffness in B-Lymphocytes 259 䊏
Fig. 1. Myo1g deficiency reduces CD44-dependent spreading and adhesion. A: Changes in cell stiffness were recorded by placing
the AFM probe onto the cell, followed by the determination of the elastic response by approaching and retracting the AFM probe
from the cell in order to obtain the force versus distance curves. B: Force vs. distance (F vs. D) curves showing the stiffness of WT
or Myo1g2/2 unstimulated (black lines) or stimulated (gray lines) cells. C: Resting B-lymphocytes or LPS plus IL-4–activated B-lym-
phocytes were allowed to adhere to poly-L-lysine coated coverslips and then analyzed with an AFM microscope. The graph shows the
cell stiffness of B-lymphocytes, derived from the analysis of 25 cells and presented as mean 6 SD. D: Activated primary mouse B-
cells, spread over a-CD44 or a-IgM, were stained with TRITC-phalloidin to detect F-actin, the arrows indicate membrane-
projections in B-lymphocytes; note that Myo1g2/2 cells did not spread as much as WT cells with a-CD44 stimuli, Scale bar pano-
ramic pictures: 60 lm, Scale bar in enlarged pictures 5 lm. E: The spreading of B-lymphocytes over either a-CD44 or a-IgM was
measured by determining their relative area. Spreading of Myo1g2/2 B-cells in IgM substrate was compared to that of WT cells. For
D, E, and F, three independent experiments were performed, and a representative experiment is shown. F: The width and the length
of spread lymphocytes were measured to determine “shape factor” values for each cell, corresponding to the coefficient of these two
parameters. A total of 12 fields for each condition were analyzed, and around 20 random cells per field were measured. Each plot
represents a single cell. G: Equal numbers of activated B-lymphocytes were placed in 96-well plates coated with a-CD44, a-IgM, or
poly-L-lysine as a control. The absorbance of each sample was determined and data plotted as the mean of three independent experi-
ments. H: Resting B-lymphocytes were placed on fibronectin-treated coverslips, and the cells incubated for 30 min and then fixed
with 4% PFA for 20 min. After fixation, the cells were permeabilized with 0.01% Triton in PBS for 10 min at 48C and then stained
with a-Myo1g for 20 min, followed by a-rabbit-Alexa488 for 20 min and a-B220-biotin plus streptavidin-Cy3. Finally, the cells
were observed by confocal microscopy. Images show four slices of adherent cells; the positions of these pictures are as follows: basal,
one-third, two-thirds, and apical region of the cell. The coefficient of polarization was determined in these cells. Scale bar 5lm. I:
The graph shows the polarization coefficient of Myo1g, derived from the analysis of 30 cells, from at least three independent experi-
ments. Values represent mean 6 SD. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
a rounded spreading pattern with an a-CD44 stimulus,
while using an IgM stimulus, the Myo1g2/2 B-cells became
elongated, similar to the WT cells; the total amount of
CD44 was similar in the two groups of B-cells (data not
show). We also compared adhesion to a-CD44 and a-IgM
to evaluate the participation of Myo1g in this phenomenon.
As shown in Fig. 1g, the adhesion of Myo1g2/2 B-cells and
WT cells to a-IgM was comparable. However, when
Myo1g2/2 B-cells were plated over a-CD44, the adhesion
of the cells was significantly reduced when compared with
WT B-cells.
To examine the cellular activities in which Myo1g is
involved, we observed the localization of Myo1g during B-
cell attachment to fibronectin-coated coverslips. Myo1g was
polarized to the site of interaction with fibronectin in a
large proportion of cells (Fig. 1h); this polarization may
indicate Myo1g activity. The mean fluorescence intensity
(MFI) observed in these interaction sites was measured,
indicating that Myo1g was polarized to the sites of contact
(Fig. 1i).
Resting B-cells have a well-defined spherical shape with
uniform actin distribution throughout the cell periphery,
but in the presence of various chemokines, such as
CXCL13, the cells switch their morphology from a spheri-
cal to a more elongated shape with membrane projections
(migratory phenotype) [Saez de Guinoa et al., 2011]. We
analyzed the migratory phenotype of the cells at different
time-points and found that Myo1g2/2 B-cells were less
prone to the migratory phenotype during the first minutes
of the response (5–10 min). Later, the cells from the WT
mice became less elongated and the differences became less
evident compared with the Myo1g2/2 B-cells, (white
arrows, Fig. 2a). A more detailed view of this process can be
seen in Fig. 2b (white arrows). To quantify these differen-
ces, we once again used the “shape factor” (Fig. 2c). To
eliminate the possibility that these changes were due to dif-
ferences in CXCR5 expression, WT and Myo1g2/2 B-cells
were stained and analyzed by FACS. The expression of
CXCR5 is similar in the B-lymphocytes from both types of
mouse [Maravillas-Montero et al., 2014].
We next evaluated whether the lack of Myo1g affects the
migration in vitro. We have previously evaluated the migra-
tion on fibronectin-coated surfaces, where we observed an
impaired migration under CXCL13 stimulation. In this
context, we performed chemotaxis assays in Transwell
chambers to evaluate the response to this chemotactic agent
in cell suspension. The results demonstrated a reduced
migration of Myo1g-deficient B-cells when compared with
WT B-lymphocytes (Fig. 2d).
To further investigate if Myo1g is located at the leading
pole of B-cells, the localization of Myo1g was determined
and compared with actin and CD44 (white arrow, Fig. 2e).
The results presented in this study showed that polarization
of CD44 was similar in B-cells from Myo1g2/2 mice com-
pared with that in WT cells (white arrow, Fig. 2f ). We used
CYTOSKELETON
a polarization index; this coefficient was calculated for each
cell presenting polarization by dividing the mean fluorescence
intensity of actin and CD44 signals at the capping site by the
mean fluorescence intensity of the rest of the cell. A polariza-
tion coefficient > 1.5 indicated that CD44 was polarized to
the capping site; the polarization index of CD44 was lower
in Myo1g2/2 compared with WT B-cells (Fig. 2g), but the
differences were not statistically significant.
To determine whether CXCR5 is altered by Myo1g defi-
ciency, we performed a migration assay to analyze the local-
ization of this chemokine receptor in migrating cells. The
data showed a delay in the polarization of this chemokine
receptor in Myo1g2/2 B-cells (white arrows, Supporting
Information Fig. 1a). We decided to determine the intracel-
lular localization of CXCR5 during migration and observed
an impaired mobilization of CXCR5 to the leading pole
(white arrows, Supporting Information Fig. 1b), suggesting
that cell stiffness is also involved in the localization of this
receptor.
Myo1g Deficiency Increases the Internalization
of Transferrin Receptor (TfR) and the
Endocytosis of Microbeads
Endocytosis of large particles requires a less rigid mem-
brane, which correlates with the reduction of cell stiffness
in Myo1g-deficient B-cells. Therefore, we evaluated the
endocytosis of soluble Tfn and compared it with the inter-
nalization of a-LFA-1 or IgG-coated microbeads by B-
lymphocytes.
Using a Tfn internalization kinetic assay, we recorded an
increase in Tfn endocytosis by Myo1g2/2 B-cells, which
became statistically significant after 30 min (Fig. 3a); this
increased endocytosis was not due to differences in the lev-
els of TfR on the cell surface (Fig. 3b). A similar phenom-
enon was observed with internalization of a-LFA-1- or
IgG-coated microbeads; the data showed increased internaliza-
tion of a-LFA-1- or IgG-coated microbeads by Myo1g2/2 B-
cells (Figs. 3c and 3d). As a whole, these results indicate that a
less rigid membrane allows an increased internalization of both
soluble and particulate ligands, indicating that Myo1g is an
active player in these processes.
Topology of the Plasma Membrane Is Affected
by Myo1g Deficiency
Class I myosins are localized to and actively involved in the
development of various types of membrane extensions in
several cell types. They are also required for the formation
and maintenance of these microstructures. Therefore, we
evaluated the presence and length of microvilli in activated
B-lymphocytes using electron microscopy. The results dem-
onstrated that the microvilli of Myo1g2/2 B-cells were
shorter and less abundant than those of WT B-cells (arrows
Figs. 4a and 4b).
Based on these data, we decided to determine the topog-
raphy of the plasma membrane more precisely. The
Myo1g Mediates Cell Stiffness in B-Lymphocytes 261 䊏
Fig. 2. Myo1g deficiency affects the dynamic changes of B-lymphocytes. A, B: Resting primary WT and Myo1g2/2 B-cells were placed on
fibronectin-covered coverslips for 30 min, after that the cells were treated with CXCL13 and incubated for 5, 10, 20 and 60 min, and then fixed
and stained with TRITC-phalloidin for 20 min, the arrows indicate the cells with migratory phenotype. The cells were then analyzed by confocal
microscopy, Scale bar panoramic pictures: 60 lm, Scale bar in enlarged pictures 5 lm. C: The “shape factor” of B-lymphocytes that adhered to
fibronectin and were stimulated with CXCL13 was evaluated. Twelve fields for each condition were analyzed, and around 20 randomly selected
cells per field were measured. The graph shows the morphology of cells with a migratory phenotype, derived from at least three independent
experiments and presented as mean 6 SD Each plot represents a single cell. D: Resting primary WT and Myo1g-deficiency B-cells were placed in
transwell chambers with CXCL13 and the cells were stained with a-B220-PB and the fluorescence was acquired by flow cytometry. E: Resting pri-
mary WT B-cells were placed on fibronectin-covered coverslips for 30 min and then treated with CXCL13 for 20 min; then, they were fixed and
stained with polyclonal a-Myo1g plus FITC-a-IgG-rabbit and a-CD44-biotin (clone IM7) plus SA-Cy3 or TRITC-phalloidin for 20 min. After
the incubation the cells were analyzed by confocal microscopy to determine Myo1g localization, arrows indicate the localization of Myo1g in the
leading pole. Scale bar 5 lm. F: Resting primary WT and Myo1g2/2 B-cells were placed on fibronectin-covered coverslips for 30 min; after that,
the cells were treated with CXCL13 several times and were fixed and stained with a-CD44-biotin (clone IM7) plus SA-Cy3 for 20 min. The cells
were then analyzed by confocal microscopy to determine CD44 polarization to the leading edge, arrows illustrate the CD44 polarization in B-
cells. Scale bar 5 lm. G: A polarization index was calculated for each cell presenting with this polarization, by dividing the mean fluorescence inten-
sity of actin and CD44 signals at the cross-link site by the mean fluorescence intensity of the rest of the cell. When capping gave a polarization coef-
ficient > 1.5, it was considered a positive result that CD44 was polarized to the cross-linking site. The graph shows the polarization index of
CD44 to the leading edge, derived from the analysis of a total of 100 cells with this structure, from at least three independent experiments, differ-
ences significantly were not observed. Values represent mean 6 SD. [Color figure can be viewed in the online issue, which is available at wiley-
onlinelibrary.com.]

䊏 262 Lopez-Ortega et al. CYTOSKELETON

Fig. 3. Myo1g deficiency increases endocytosis of Tfn and receptor-dependent phagocytosis in B-lymphocytes. A: Resting B-
lymphocytes were treated with FITC-Tfn for 1 h at 48C and were then incubated at 378C for several different durations. After incu-
bation, the fluorescence was quenched by the addition of 0.5% trypan blue for 30 s, and the cells were extensively washed with 1x
PBS. Finally, the cells were fixed and fluorescence was analyzed by flow cytometry. The graph shows the percentage of Tfn endocy-
tosed by B-lymphocytes, derived from the analysis of six independent experiments and presented as mean 6 SD. B: TfR on plasma
membrane of B-lymphocytes. Cell surface TfR of resting or LPS plus IL4-activated B lymphocytes from WT or Myo1g-deficient B-
lymphocytes were stained with goat a-mouse TfR during 20 min, then, the cells were treated with FITC-rabbit a-goat IgG and ana-
lyzed by flow cytometry. The graph shows the amount of TfR on the surface derived from the analysis of four independent experi-
ments, values are mean 6 S.D. C: Resting B-lymphocytes were treated with 0.5 lm a-LFA-1 coated-microspheres for 30 min at
378C, after which the fluorescence was quenched by the addition of 0.5% trypan blue for 30 s and the cells extensively washed with
1x PBS. Finally, the cells were fixed and the fluorescence was analyzed by flow cytometry. D: The graph shows the MFI of microbe-
ads inside Myo1g2/2 or WT B-cells; the data are derived from five independent assays, values represent mean 6 SD.

abundance of several plasma membrane structures in
Myo1g2/2 B-cells was indeed lower than in WT B-cells
(Fig. 4c). We also found smooth areas on Myo1g2/2 B-
cells (red arrows, Fig. 4c). A more detailed view of the
membrane topography can be seen in Fig. 4d. In this con-
text, we used a roughness index; this index is a coefficient
that indicates the cell surface texture. We quantified the
roughness of the plasma membrane and the height of mem-
brane protrusions at the cell surface. The data showed that
the membrane was smoother (Fig. 4e) and microvilli were
shorter on Myo1g2/2 B-cells (Fig. 4f ). These values could
indicate cell surface alterations in the boundary between the
actin-cytoskeleton and the plasma membrane.
In summary, we determined that the class I myosin,
Myo1g is an active player in the maintenance of B-
lymphocyte cell stiffness, which is directly linked to endocy-
tosis and the development of membrane projections.
Further work is necessary to elucidate the mechanisms by
which Myo1g modulates these processes, as well as to deter-
mine the reason the Myo1g deficiency did not have a more
pronounced effect on cell stiffness.
Discussion
In the current study, the effect of Myo1g deficiency on cell
stiffness was assessed to determine whether its reduction

CYTOSKELETON Myo1g Mediates Cell Stiffness in B-Lymphocytes 263 䊏

Fig. 4. Myo1g deficiency affects plasma membrane topology. A: Activated WT and Myo1g2/2 B-lymphocytes were fixed with
2.5% glutaraldehyde for 1 h and then processed for scanning electron microscopy. The images show the presence of membrane pro-
trusions such as microvilli, arrows show membrane-projections of B-cells. Scale bar 5 lm. B: The graph shows the length of micro-
villi in these cells, derived from the analysis of a total of 100 cells from at least three independent experiments and presented as
mean 6 SD. C, D: Activated B-lymphocytes were fixed with 4% paraformaldehyde for 20 min and processed for AFM (as described
above). The pictures show the topography of the cell surface of activated WT and Myo1g2/2 B-cells; arrows illustrate the presence of
smooth areas in Myo1g2/2 B-cells in C), Scale bar 2 lm. The graphs show the roughness E) and height of membrane-protrusions
F) on the surface of wild-type or Myo1g2/2 B-cells, derived from the analysis of 30 cells, from at least three independent experi-
ments. Values represent mean 6 SD. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

impairs B-cell functions such as endocytosis and spreading.
The results obtained here are in agreement with reports by
Olety et al. [2010] [Olety et al., 2010] and Gerard et al.
[2014] [Gerard et al., 2014] who observed a decrease in
elasticity in cells with reduced or depleted expression of
Myo1g; the Jurkat cell line [Olety et al., 2010] and primary
T-lymphocytes [Gerard et al., 2014], respectively, were used
for these studies.
The increase in these biophysical parameters in LPS-
activated B-lymphocytes has been observed in various cell
types such as macrophages and microglial cells [Pontes
et al., 2013]. In an attempt to explain this increase in cell
stiffness, we hypothesized that a possible change in the loca-
tion or posttranslational modifications of Myo1g, alone or
in combination with other elements, may contribute to the
increase in cell rigidity. Previous data from literature shows
the same expression of class I myosins (Myo1c [Yip et al.,
2008], and Myo1e [Wenzel et al., 2015] during activation,
but changes in the location of these molecules after stimula-
tion have been reported; an example is the association of
Myo1c to vesicles after the treatment of adipocytes with
insulin [Yip et al., 2008]. Thus, we verified the expression
of Myo1g under resting and activated conditions where we
observed a similar expression of this protein in B

䊏 264 Lopez-Ortega et al. CYTOSKELETON

lymphocytes (Supporting Information Fig. 2). Therefore,
more studies on the mechanisms controlling cell stiffness
during cell activation are needed to clarify these processes.
A reduction in cell spreading has also been reported in
other cell types involving different class I myosins [Santos-
Argumedo et al., 2013]; for example, Myo1c is implicated
in the spreading of B-cells [Maravillas-Montero et al.,
2011], HeLa cells [Brandstaetter et al., 2012], and fibro-
blasts [Oh et al., 2013; Santos-Argumedo et al., 2013].
These differences between cell spreading on a-IgM and a-
CD44 may be due to changes in cell stiffness. This is sup-
ported by a report in which B-lymphocytes treated with
a-IgM displayed increased membrane rigidity, driving the
recovery of membrane tension [Krolick et al., 1977; Paster-
nak and Elson, 1985]. The absence of defects seen in cell
spreading induced via a-IgM can be explained by the com-
pensatory role played by other class I myosins (such
as Myo1c and Myo1e that are also expressed in B-cells)
[Maravillas-Montero et al., 2011].
Changes in cell rigidity can affect migration; for example,
an increase in membrane tension leads to inhibition of lead-
ing edge formation in neutrophils and a decrease in leading
edge activities [Houk et al., 2012]. Changes in membrane
stiffness oppose the development of membrane protrusions;
therefore, it is expected that an increase in membrane ten-
sion would inhibit lamellipodium extension in migratory
cells. This idea has been confirmed experimentally in fibro-
blasts [Raucher and Sheetz, 2000]. Furthermore, an
increase in membrane tension inhibits the development of
membrane protrusions during cell spreading in fibroblasts
[Gauthier et al., 2011].
In contrast, when we analyzed endocytosis, we observed
an increase in this phenomenon; these results are in agree-
ment with those reported by Marion et al. [2005] that dem-
onstrated that overexpression of myosin 1B in Entamoeba
histolytica produced a decrease in phagocytosis [Marion
et al., 2005], which was attributed to an increase in cortical
tension. Overexpression of several class I myosins in D. dis-
coideum has been associated with an increase in phagocyto-
sis and membrane tension [Dai et al., 1999; Nambiar et al.,
2009]. Therefore, as stated previously [Keren, 2011], a
reduction in membrane tension is expected to increase
endocytic processes. In contrast, it is also known that this
same alteration concurrently reduces the exocytosis of GPI-
anchored proteins in HeLa cells [Gauthier et al., 2009].
Additionally, we have previously reported an increase in the
phagocytosis of S. aureus and a reduction in the secretion of
tumor necrosis factor-a by Myo1g2/2 B-cells [Maravillas-
Montero et al., 2014].
Finally, the formation of membrane extensions such as
pseudopods, filopodia, and microvilli is dependent on
membrane tension. In this study, we demonstrated that
Myo1g deficiency affects the length of these microstruc-
tures, indicating that this protein is involved in maintaining
cell stiffness in B-cells. Pietuch et al. [2013] demonstrated
CYTOSKELETON
that a lower membrane tension resulted in shorter mem-
brane projections [Pietuch et al., 2013]. Additionally,
Hecht et al. [2011] showed that cells that have an increased
rate of endocytosis have fewer microvilli on their surface
[Hecht et al., 2011].
The roughness of normal cells indicates an adequate link
between the cytoskeleton and the plasma membrane [Gira-
sole et al., 2007]. Cell surface roughness is relevant because
it is implicated in several cell processes such as migration,
the regulation of intracellular contacts, and cell adhesion
[Keren et al., 2008; Krobath, 2007]. As a whole, the results
reported in this paper provide evidence of the participation
of Myo1g in maintaining cell stiffness, and show how this
property of the membrane affects various cellular functions
in B-lymphocytes.
Materials and Methods
Mice and Reagents
Female C57BL/6J (WT) or Myo1g-deficient (Myo1g2/2,
C57BL/6J background) mice (8–12 weeks old) described in
[Maravillas-Montero et al., 2014], were used in all experi-
ments. The mice were reared at the Centro de Investigacion
y de Estudios Avanzados (Mexico City, Mexico) animal
facility, and the Animal Care and Use Committee of Centro
de Investigacion y de Estudios Avanzados approved all
experiments. The following is a list of the antibodies and
reagents used: purified rabbit polyclonal immunoglobulin
(Ig)G a-Myo1g [Patino-Lopez et al., 2010]; NIM-R8 (a-
CD44) [Santos-Argumedo et al., 1997]; a-rat-PE (BD
Pharmingen, San Diego, CA); purified a-goat-PE; a-IgG-
rabbit-Alexa488 (Molecular Probes, Eugene, OR, USA); a-
IgG-rabbit-FITC (Jackson ImmunoResearch, West Grove,
PA, USA); TRITC-phalloidin (Molecular Probes); FITC-
phalloidin (Sigma Chemical Co, St Louis, MO, USA);
Pacific Blue a-B220 (BD Pharmigen); transferrin (Tfn)
(Sigma Chemical Co); fibronectin (2.5 lg/mL, Takara Bio
Inc., Otsu-shi, Shiga, JP); recombinant mouse CXCL13
(PeproTech, Offenbach, Germany); and biotinylated mouse
a-CXCR5 (BD Pharmigen).
B-Lymphocyte Isolation and Activation
Mononuclear cells were isolated from spleen tissue by Ficoll
density gradient separation. B-cells were enriched by pan-
ning using plastic dishes coated with anti-Thy-1 ascites
(monoclonal NIM-R1 antibodies, a-CD90). For activa-
tion, 2 3 106 cells were incubated in 1 mL 10% fetal
bovine serum (FBS) supplemented with Roswell Park
Memorial Institute (RPMI) 1640 (Life Technologies) con-
taining lipopolysaccharides (LPS) from Escherichia coli
O55:B5 at 50 mg/mL (Sigma) plus 10 U/mL IL-4 (R&D
Systems, Minneapolis, MN, USA) for 48 h at 378C.
Myo1g Mediates Cell Stiffness in B-Lymphocytes 265 䊏
Measurement of Cell Stiffness
To obtain the cell constant we considered the system
depicted in Fig. 1a which corresponds to two springs in
series, one was the cantilever with spring constant and the
other was the cell with spring constant giving as a result
the apparent constant that can be seen in the Fig. 1b. The
KcKa
measured membrane constant is thus given by Km5 Kc2Ka
since kc > km the change in the apparent constant (slope)
reflects the change in cell stiffness. The experimental F vs.
D curves were obtained for each set of cells in each of the
categories of this work (Unstimulated Wild Type, Unstimu-
lated Myo1g2/2, Stimulated Wild Type and Stimulated
Myo1g2/2). A sample of one of these measurements is pre-
sented in Fig. 1b. These curves are nearly linear and the
increase in slope comes from an increment in the cell stiff-
ness (see Ovalle-Garcıa et al., 2011 for a more general
model) [Ovalle-Garcia et al., 2011].
To select the region to be sampled, first we took a 2D
image of the cell surface using the contact mode of the
AFM by placing the probe on top of the cell under micro-
scope (X20) observation. If the cell surface showed a hill
shape after the scan, 16 force vs distance (F vs. D) curves
were acquired along a 1 mm line at the top of each cell. All
AFM measurements were performed in a liquid environ-
ment at 298C. In order to have well-behaved F vs. D curves,
the vertical displacement of the probe was adjusted and the
force-resistance was recorded. Each curve had 500 (force-
distance) ordered pairs. The average of the 16 curves was
used to obtain the cell stiffness. The vertical probe displace-
ment was 1 mm on average, applied with a 0.2 Hz fre-
quency, and 200 nm of that displacement was used for
determination. The AFM was equipped with the software
PROSCAN 1.7. Si3N4 cantilevers with nominal spring
constant of k 5 30 mN/m and 728 pyramidal tip with
nominal radius of 53 nm were used (both, software and
cantilevers, were from TM Microscopes). The cantilevers
were calibrated using the Torii et al., 1996 procedure [Torii
et al., 1996].
Analysis of Cell Topography
Specific features in the AFM topography were analyzed by
line profiling routines provided in the WSXM imaging soft-
ware (www.nanotec.es). The heights and roughness of
raised regions were measured relative to the surrounding
membrane, not the substrate. Because the raised domains
were irregularly shaped, care was taken for each zone in
acquiring representative profiles. The profiles were then
analyzed using the WSXM imaging software to obtain sev-
eral graphs and values of roughness and height. All the rep-
resentative height and roughness data were entered into
GraphPad for statistical analysis (www.graphpad.com/scien-
tific-software/prism).
䊏 266 Lopez-Ortega et al.
Scanning Electron Microscopy
Cells were fixed with 2% (v/v) glutaraldehyde in 0.1 M cac-
odylate buffer, pH 7.2, and dehydrated with increasing
concentrations of ethanol. Samples were critically point-
dried in a Samdri apparatus, gold coated with an ion-
sputtering device (Jeol-JFC-1100), and examined with a
Jeol JSM-7100F field emission scanning electron micro-
scope. In the image, we measured the length (from bottom
to top) of each microvillus, through the option of straight
line for straight microvilli and for curved microvilli, we
used the Bezier curve tool option.
Spreading Assays
Glass slides were coated with 20 lg/mL of NIM-R8 or a-
IgM in phosphate-buffered saline (PBS) for 1 h at 378C.
The coverslips were blocked with PBS containing 10% fetal
calf serum for 1 h at 378C and then washed thoroughly
with medium before use. Activated B-cells were transferred
without washing to the pre-coated glass slides and incu-
bated for 1 h at 378C. The cells were then fixed with 4%
paraformaldehyde for 15 min, washed again with PBS, and
permeabilized with 0.1% Triton X-100 in PBS for 10 min.
After the incubation, the cells were washed with PBS,
treated with TRITC-phalloidin, and mounted as described
previously [Maravillas-Montero et al., 2011]. The obtained
slides were investigated using an FV300 Olympus micro-
scope using 603 objectives and NIH ImageJ for analysis
(http://rsbweb.nih.gov/ij).
Adhesion Assays
Polystyrene plates (96-well, Nalge Nunc International) were
coated with various substrate molecules such as poly-L-
lysine (0.01%, Sigma), fibronectin (2.5 lg/mL, Takara),
and purified a-CD44 or a-LFA-1, for 1 h at 378C. The
plates were then washed twice with PBS before adding 4 3
105 panning-enriched B-cells in 200 lL of RPMI 1640 per
well. The cells were allowed to adhere for 1 h at 378C. The
plates were then washed with 300 lL of PBS and fixed with
4% paraformaldehyde for 10 min before adding Crystal
Violet (Crystal Violet 7.5 g/L, NaCl 2.5 g/L, formaldehyde
1.57%, methanol 50%) for an additional 5 min. The cells
were washed thoroughly three times with distilled water
and solubilized with 10% sodium dodecyl sulfate, and the
plates were read at 540 nm using a Multiskan Ascent plate
reader (Thermo Scientific, Waltham, MA, USA). After sub-
traction of nonspecific colorimetric readings to obtain the
absolute binding, the absorbance for each well was regis-
tered in at least four wells per condition in three independ-
ent experiments.
Migration Assay
We used a Zigmond chamber (Neuro Probe, Inc., Gaithers-
burg, MD, USA) for the quantification of migration.
Briefly, 1 3 106 B-lymphocytes were suspended in 0.5 mL
CYTOSKELETON
of 10% FBS supplemented with RPMI 1640 (Life Technol-
ogies, Carlsbad, CA, USA) and immediately plated onto
glass coverslips that had previously been coated with 2.5
lg/mL of fibronectin (Takara). The coverslips were then
incubated for 30 min at 378C in 5% CO2 to allow the cells
to attach. The coverslips, with the cells attached, were
gently washed with 2.0 mL PBS, and 100 lL of supple-
mented RPMI 1640 was pipetted onto the coverslips,
which in turn were placed upside-down onto the Zigmond
slides. One of the grooves in the Zigmond chamber was
filled with supplemented medium (80 lL) and the cham-
ber was placed under a microscope. A baseline image was
obtained at 403 magnification and the other groove was
then filled with CXCL13 (50 lg), also dissolved in supple-
mented medium. Digital images of the cells were taken
every 30 s for 1.5 h maintaining the temperature of the
room at 35–398C. For analyzing the trajectories and speed
of migration, the migration tracks were traced for at least
100 lymphocytes of each genotype, in six independent
experiments, using NIH ImageJ software with chemotaxis
and migration tool 2.0 (Ibidi).
Internalization Assays
For in vitro assessment of phagocytosis, panning-enriched
B-cells were incubated (1 3 106 cells per well) with
antibody-coated 0.5-mm polystyrene fluorescent micro-
spheres (Molecular Probes). The bead-to-cell ratio was 10:1
and the beads were placed in 24-well plates in 200 lL of
supplemented RPMI 1640 medium for 1 h at 378C in 5%
CO2. The cells were then harvested and washed with PBS
before adding 0.5 mL of a Trypan Blue solution (50 mg/
mL) to quench the fluorescence of un-ingested particles by
flow cytometry assays. After a brief incubation of 2 min,
the dye was washed with PBS three times, and the cells
were fixed with 3.7% formaldehyde and stained with Pacific
Blue a-B220. Cell suspensions were then analyzed using a
CyAn ADP flow cytometer (Beckman Coulter, Inc., Brea,
CA, USA); the flow cytometry data were analyzed using
FlowJo software.
Transferrin Endocytosis Assay
The cells were incubated with Tfn–FITC for 60 min at
48C and then incubated at 378C for various periods before
fixation. Levels of cell-associated Tfn–FITC were deter-
mined by fluorescence-activated cell sorting (FACS) analysis
using a CyAn ADP flow cytometer (Beckman Coulter); the
flow cytometry data were analyzed using FlowJo software.
Chemotaxis Assay
The chemotaxis of B-cells to the chemoattractant CXCL13
was investigated in 5-lm pore size Transwell plates (Corn-
ing). Briefly, a total of 5 3 105 B-lymphocytes were placed
in 500 lL of RPMI containing 10% FBS, and the cells
were added to the upper chamber of the Transwell plates.
CYTOSKELETON
CXCL13 was added to the lower wells. The plates were
incubated for 3 h at 378C in 5% CO2. Controls without
CXCL13 (bovine serum albumin) were maintained in each
experiment to account for passive diffusion of cells. The
migration of B-lymphocytes was determined by counting
the cells that migrated to the lower chamber as a percentage
of the total cells that were loaded, with the passive diffusion
subtracted as background.
Statistical Analysis
Results are presented as mean 6 standard deviation (SD).
An unpaired two-tailed Student’s t-test was used to assess
the statistical significance of differences between the WT
and Myo1g2/2 groups. The p-values obtained and the
number of samples or cells (n) used are stated in each figure
legend.
Acknowledgments
The authors gratefully thank Dr. Steve Shaw from NCI,
NIH for providing Myo1g2/2 mice, Dr. Hector Romero-
Ramırez for his help at different stages of this work, and
M.V.Z Ricardo Gaxiola Centeno and Tec. Victor Manuel
Garcıa Gomez for their excellent technical assistance in the
animal facility.
This work was supported by CONACyT (grant 153733)
and UNAM-DGAPA-PAPIIT IG100513. O.L.-O. was sup-
ported by fellowship 219624 from CONACyT.
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