Measuring gas vesicle dimensions by electron
microscopy
Przemysław Dutka
1
, Dina Malounda
1
, Lauren Ann Metskas
2
, Songye Chen
2,3
, Robert C.
Hurt
2
, George J. Lu
2,5
, Grant J. Jensen
2,4,*
, and Mikhail G. Shapiro
1,*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
2
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125
3
Beckman Institute, California Institute of Technology, Pasadena, CA 91125
4
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602
5
Present address: Department of Bioengineering, Rice University, Houston, TX 77030
*
Correspondence should be addressed to G.J.J. (jensen@caltech.edu) or M.G.S. (mikhail@caltech.edu)
ABSTRACT
Gas vesicles (GVs) are cylindrical or spindle-shaped protein nanostructures filled with air and used for flotation by various
cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to
their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The
diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells,
as well as their properties in imaging applications. Despite its importance, reported diameters for the same types of GV differ
depending on the method used for its assessment. Here, we provide an explanation for these discrepancies and utilize electron
microscopy (EM) techniques to accurately estimate the diameter of the most commonly studied types of GVs. We show that
during air drying on the EM grid, GVs flatten, leading to a ~1.5-fold increase in their apparent diameter. We demonstrate that
GVs’ diameter can be accurately determined by direct measurements from cryo-EM samples or alternatively indirectly derived
from widths of flat collapsed and negatively stained GVs. Our findings help explain the inconsistency in previously reported
data and provide accurate methods to measure GV dimensions.
INTRODUCTION
Gas Vesicles (GVs) are hollow, gas-filled protein nanostruc-
tures natively expressed in certain types of cyanobacteria, het-
erotrophic bacteria, and Archaea as a buoyancy aid (Walsby
1994). Recently, it was discovered that the unique physical
properties of GVs enable them to serve as genetically encod-
able contrast agents for ultrasound and other imaging methods,
allowing deep tissue imaging of cellular function (Shapiro,
Goodwill, et al. 2014; Shapiro, Ramirez, et al. 2014; Bour-
deau et al. 2018; Lu et al. 2018; Farhadi et al. 2019, 2020;
Lakshmanan et al. 2020). In addition, GVs are being applied
to acoustic manipulation and therapeutic uses of engineered
cells (Bar-Zion et al. 2019; Wu et al. 2019).
Fully formed GVs adopt two predominant shapes - cylin-
ders with conical ends or spindle-like. The GVs may be 0.1-
2
μ
m in length, or even longer when heterologously expressed
in more spacious mammalian cells (Farhadi et al. 2019). The
mean diameter of GVs isolated from different species widely
varies, but is relatively constant for the same type of GV. There
is an inverse correlation between diameter and critical col-
lapse pressure (Hayes and Walsby 1986). This correlation has
important evolutionary consequences. While wider GVs can
provide buoyancy at a lower energetic cost, they collapse at
lower pressure. This is perhaps best reflected by analyzing the
widths and collapse pressure of GVs isolated from
Planctronix
spp.
from nordic lakes of different depths (Beard et al. 1999,
2000). Three types of GVs isolated from
Planctronix spp.
had
widths of approximately 51, 58, and 67 nm with respective
collapse pressures of 1.1, 0.9, and 0.7 MPa, allowing them to
adapt to the hydrostatic pressure in different lakes (Beard et
al. 2000; Dunton and Walsby 2005).
Despite the importance of GVs’ diameter for their biophys-
ical properties, there are significant discrepancies in values
reported in the literature. For example, the width of GVs
from
Anabaena flos-aquae
(Ana) measured inside cells by
thin-section electron microscopy (EM) was approximately 70
nm (Walsby 1971), which is considerably smaller than the
value obtained by negative stain EM (ns-EM) for isolated
GVs - 136 nm (Lakshmanan et al. 2017). Similar discrepan-
cies can be observed for GVs from
Halobacterium salinarum
(Halo), whose reported values range from 45 nm to 250 nm
(Simon 1981; Offner et al. 1998; Pfeifer 2012; Lakshmanan
et al. 2017). To some extent, these discrepancies could be
explained by natural variability in diameter. However, anal-
.
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ysis of width distributions for GVs from several species of
Cyanobacteria (Hayes and Walsby 1986) or
Bacillus mega-
terium
(Mega) (Farhadi et al. 2018) shows a narrow range.
This inconsistency in diameter measurement was investigated
almost 50 years ago by Walsby (Walsby 1971). He observed
that Ana GVs have a constant width of 70 nm when mea-
sured inside cells by thin-section EM, which was close to the
value measured for the purified sample imaged using a freeze-
etching technique (75 nm). In contrast, estimations by ns-EM
ranged from 70 to 114 nm (Walsby 1971). He suggested
that the stain used in EM leads to swelling of GVs, which
increases their diameter but has little effect on the length. As
an alternative approach for assessing GV diameter, Walsby
proposed indirect measurement based on the widths of flat
collapsed GVs. The diameter of Ana GVs measured using
this strategy was approximately 85 nm (Walsby and Bleything
1988). Archer and King gave another potential explanation
for discrepancies in GV measurements. They proposed that
the isolation process leads to deformations, increasing the
width of GVs (Archer and King 1984). Regardless of these
concerns, the diameter of GVs has been routinely assessed for
isolated specimens by ns-EM.
As GVs have attracted more attention in biotechnology
applications, accurate estimates of their diameter have become
a critical input into GV engineering. For that reason, we
investigated the discrepancies in reported GV diameters using
modern microscopy tools. Using these updated techniques,
we provide measurements for the most commonly studied
GVs: Ana, Mega, and Halo. For Halo, we analyzed two
different GV types, which are products of the independent
gene clusters p-vac and c-vac.
RESULTS AND DISCUSSION
To more closely evaluate the behavior of stained and air-dried
GVs on the EM grid, we collected projection images for
different types of GVs at 0
°
and 50
°
tilt and analyzed their
morphology
(Figure 1a and 1b)
. Although we predicted
some degree of distortions to the cylindrical shape of GVs,
the observed differences were unexpectedly large. For Ana
GVs, there was an average of 55 nm width difference between
measurements at these two angles. The pattern was similar for
both Mega and Halo GVs, although to a different degree. This
data indicates that all types of GVs flatten during the staining
procedure, adopting an elliptic cylinder shape.
Certain limitations of the ns-EM technology, such as spec-
imen flattening or stain thickness irreproducibility, were pre-
viously described (Frank 2006). However, the observed de-
formation of the GV protein shell is not like the typical flat-
tening reported before, where sample was mainly affected
in z-direction with little to no effect on the x.y-dimensions
(Frank 2006). Since GVs produce strong contrast on EM
even without staining, we decided to take advantage of this
unique property and evaluate the effect of the stain. Analysis
of unstained, air-dried Ana GVs samples at 0
°
and 50
°
tilts
show on average 20 nm difference in diameter
(Figure 1c)
,
Figure 1.
GVs flattening on the EM grid.
(a) Schematic
showing cross-section of the flattened GV at 0° and 50° tilt.
(a,b) Representative projection images at 0° and 50° tilt for
(b) negatively stained and air-dried Ana, Mega, and Halo GV;
and (c) unstained, air-dried Ana GV. Scale bar, 200 nm.
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Figure 2.
Diameter measurement for Mega, Ana, and Halo GVs.
(a) Representative cryo-EM of intact GVs used for
direct diameter measurement. (b) Representative ns-EM images of collapsed GVs used for indirect diameter assessment based
on widths of flat collapsed regions. Scale bar, 100 nm. (c) Diameter distribution for Mega, Ana, and Halo GVs measured by
cryo-EM and collapsed ns-EM. Center line indicates median, the box limits denote the interquartile range and the whiskers
absolute range. Each dot represents an individual measurement.
which is significantly less than the stained sample, but not
negligible.
Distortions to the GV shape are the effect of the unique
mechanical properties of GVs’ protein shell. In ns-EM, the
sample lies on a carbon support; thus, we suspect that GVs are
compressed by the surface tension of evaporating water. No-
tably, the degree of deformation appears to be correlated with
critical collapse pressure. Halo GVs, which experience the
most flattening, are also the least robust among investigated
GVs, with collapse pressure of 0.1 MPa (Lakshmanan et al.
2017). In contrast, Mega GVs, which have a much higher col-
lapse pressure of 0.7 MPa (Lakshmanan et al. 2017), flatten
the least.
To obtain more accurate measurements of GV diameter,
we used two complementary methods. First, we imaged the
GVs with cryo-EM, which preserves GVs’ cylindrical shape.
Unfortunately, cryo-EM is a more demanding technique, re-
quiring time-consuming sample optimization, larger sample
quantities, and access to a more sophisticated instrument. Al-
ternatively, we inferred GV diameter from the widths of flat
collapsed GVs with negative staining, as measured by Walsby
and Bleything (Walsby and Bleything 1988). This method,
which equates the collapsed GV width with half of the intact
cylindrical circumference, should allow for a faster and more
accessible estimation of GV dimensions. We decided to ana-
lyze diameter distribution for Mega, Ana, and Halo GVs using
both strategies. Cryo-EM of intact GVs and collapsed ns-GV
imaging resulted in similar values for each analyzed GV type
(Figure 2, Table 1)
, with differences within statistical error.
Mega and Ana GVs appear to have a uniform diameter, vary-
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Table 1.
Measured diameters (mean ± s.d.) for Mega, Ana,
and Halo GVs by EM
GV type
Intact GVs
(ns-EM)* (nm)
Intact GVs
(cryo-EM)
(nm)
Collapsed GVs
(ns-EM) (nm)
Mega
73 ± 14
52 ± 6
54 ± 5
Ana
136 ± 21
85 ± 4
89 ± 6
Halo (c-vac)
251 ± 51
111 ± 32
116 ± 21
Halo (p-vac)
182 ± 22
171 ± 19
*Previously reported by Lakshmanan et al. (2017).
ing within a narrow range
(Figure 2c, Table 1)
. In contrast,
Halo GV diameters varied. Halo is capable of producing two
types of GVs. Spindle-shaped GVs are encoded by the p-vac
gene cluster located on an endogenous plasmid, while the
c-vac cluster located on a mini-chromosome generates cylin-
drical GVs (Pfeifer 2012). According to our measurements,
the diameter of both types of Halo GVs varies
(Figure 2c)
.
However, some of this variability may be due to imperfect
classification. All GV types begin their assembly as bicones,
which look like smaller spindle-shape p-vac Halo GVs (Pfeifer
2012). Thus, some c-vac GVs, in their bicone phase, could
have been classified as p-vac GVs. This misclassification
could have made a minor contribution to the overall diameter
distribution. Overall, the range of diameter values for differ-
ent GV types suggest that Ana and Mega GVs have tighter
regulation over diameter compared to Halo GVs. However,
it is not yet known what the physiological consequences of
this regulation are or how exactly the diameter is adjusted in
growing GVs.
Taken together, our findings provide an explanation for dis-
crepancies in previous GV diameter measurements reported
in the literature. Although ns-EM is routinely used to evaluate
the morphology and dimensions of intact GVs (Li and Cannon
1998; Ramsay et al. 2011; Xu et al. 2014; Lakshmanan et al.
2017; Farhadi et al. 2018), our data show that this method
causes GV flattening and inaccurate apparent diameter. In-
stead, cryo-EM of intact GVs and ns-EM of flat collapsed
GVs provide correct dimensions that are mutually consistent
between the two methods, as shown here for three commonly
studied GVs variants.
MATERIALS AND METHODS
GV expression and purification
GVs were either isolated from native sources (Ana and Halo)
or expressed heterologously in Rosetta 2(DE3) pLysS
E.coli
cells (Mega) as previously described (Lakshmanan et al. 2017).
In the final two or three rounds of buoyancy purification, sam-
ple buffer was exchanged to 10 mM HEPES, pH 7.5. Con-
centrations were measured by optical density (OD) at 500
nm using a spectrophotometer (NanoDrop ND-1000, Thermo
Scientific).
Negative stain electron microscopy
For imaging of intact GVs, the purified sample was diluted
to OD
5
00
~0.5 for Ana and Halo, and OD
5
00
~0.2 for Mega.
When data for collapsed GVs were collected, diluted sam-
ples were squeezed in a sealed syringe until turned transpar-
ent. 3
μ
L of the target sample was applied to a freshly glow-
discharged (Pelco EasiGlow, 15mA, 1 min) Formvar/carbon-
coated, 200 mesh copper grid (Ted Pella) for 1 min before
blotting. Afterward, the sample was incubated for 1 min with
a 0.75% uranyl formate solution before blotting and air-dried.
Image acquisition was performed using a Tecnai T12 (FEI,
now Thermo Fisher Scientific) electron microscope at 120 kV,
equipped with a Gatan Ultrascan 2k X 2k CCD.
Cryo-electron microscopy
For cryo-EM, Quantifoil R2/2 200 Mesh, extra thick car-
bon, copper grids (EMS) were glow discharged (Pelco Eas-
iGlow, 10mA, 1 min). Freshly purified Mega (OD
5
00
~1), Ana
(OD
5
00
~15), and Halo (OD
5
00
~8) GVs sample was frozen
using a Mark IV Vitrobot (FEI, now Thermo Fisher Scientific)
(4
°
C, 100% humidity, blot force 3, blot time 4s). Micrographs
were collected on a 300kV Titan Krios microscope (FEI, now
Thermo Fisher Scientific) with an energy filter (Gatan) and
equipped with a K3 6k x 4k direct electron detector (Gatan).
Data were collected using SerialEM software with a pixel size
of either 1.4 Å (x64,000 magnification) or 2.15Å (x42,000
magnification) and -2.5 μm defocus (Mastronarde 2005).
Diameter measurement
All measurements were made using IMOD software (Kremer,
Mastronarde, and McIntosh 1996). The cylinder/spindle di-
ameter direct measurements from cryo-EM micrographs were
performed only once for each GVs at its widest region. Indi-
rectly diameter was calculated as
2
w
/
π
, where
w
is the width
of the flat collapsed gas vesicle measured from the ns-EM mi-
crograph. Sample from at least two independent preparations
were used for each measurement.
ACKNOWLEDGEMENTS
This work was supported by the National Institutes of Health
(grant R35-GM122588 to G.J.J. and R01-EB018975 to M.G.S.)
and the Caltech Center for Environmental Microbial Interac-
tions (CEMI). Electron microscopy was performed in the
Beckman Institute Resource Center for Transmission Electron
Microscopy at Caltech. Related research in the Shapiro Lab-
oratory is also supported by the Heritage Medical Research
Institute, the Pew Scholarship in the Biomedical Sciences, and
the Packard Fellowship for Science and Engineering.
AUTHOR CONTRIBUTIONS
Przemysław Dutka:
Conceptualization; methodology; in-
vestigation; formal analysis; visualization; writing – origi-
nal draft preparation; writing – review editing.
Dina Mal-
ounda:
Investigation.
Lauren Ann Metskas:
Investigation.
Songye Chen:
Investigation.
Robert C. Hurt:
Investigation.
George J. Lu:
Investigation.
Grant J. Jensen:
Concep-
tualization; writing – review editing; supervision; funding
4/6
.
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available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
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;
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acquisition.
Mikhail G. Shapiro:
Conceptualization; writing
– review editing; supervision; funding acquisition.
COMPETING INTERESTS
The authors declare no competing interests.
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