Glycobiology vol. 23 no. 2 pp. 222–231, 2013
doi:10.1093/glycob/cws149
Advance Access publication on October 19, 2012
Hyaluronan in cytosol—Microinjection-based probing of its
existence and suggested functions
Hanna Siiskonen1,2, Kirsi Rilla2, Riikka Kärnä2,
Genevieve Bart2, Wei Jing3, Michael F Haller3,
Paul L DeAngelis3,4, Raija H Tammi2,
and Markku I Tammi2
2
Institute of Biomedicine/Anatomy, School of Medicine, University of
Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland; 3Hyalose, LLC,
655 Research Parkway, Suite 525, Oklahoma City, OK 73104, USA; and
4
Department of Biochemistry & Molecular Biology, University of Oklahoma
Health Sciences Center, 940 S.L. Young Blvd., Oklahoma City, OK 73126,
USA
Received on August 15, 2012; revised on October 16, 2012; accepted on
October 16, 2012
Hyaluronan (HA) is a large glycosaminoglycan produced
by hyaluronan synthases (HAS), enzymes normally active
at plasma membrane. While HA is delivered into the
extracellular space, intracellular HA is also seen, mostly in
vesicular structures, but there are also reports on its presence in the cytosol and specific locations and functions
there. We probed the possibility of HA localization and
functions in cytosol by microinjecting fluorescent HA
binding complex (fHABC), HA fragments and hyaluronidase (HYAL) into cytosol. Microinjection of fHABC did
not reveal HA-specific intracellular binding sites. Likewise,
specific cytosolic binding sites for HA were not detected,
as microinjected fluorescent HA composed of 4–8 monosaccharide units (HA4–HA8) were evenly distributed throughout the cells, including the nucleus, but
excluded from membrane-bound organelles. The largest
HA tested (HA120 or 25 kDa) did not enter the
nucleus, and HA10-HA28 were progressively excluded
from parts of nuclei resembling nucleoli. In contrast,
HA oligosaccharides endocytosed from medium remained
in vesicular compartments. The activity of HA synthesis
was estimated by measuring the HA coat on green fluorescent protein (GFP)-HAS3-transfected MCF-7 cells.
Microinjection of HA4 reduced coat size at 4 h, but
increased at 24 h after injection, while larger HA-oligosaccharides and HYAL had no influence. As a positive
control, microinjection of glucose increased coat size. In
summary, no evidence for the presence or function of HA
in cytosol was obtained. Also, the synthesis of HA and the
active site of HAS were not accessible to competition,
1
To whom correspondence should be addressed: Tel: +358-40-5925899;
Fax: +358-17-163032; e-mail: hanna.siiskonen@uef.fi
binding and degradation by cytosolic effectors, while synthesis responded to increased substrate supply.
Keywords: hyaluronan / hyaluronan oligosaccharide /
hyaluronan synthase / MCF-7 / microinjection
Introduction
Hyaluronan (HA) is a large linear polysaccharide secreted
into extracellular matrix where it influences many important
tissue functions in health and disease, especially organ development, inflammation and tumorigenesis (Sironen et al.
2011). However, there are several reports suggesting that HA
within the cells has also specific functions.
First, there are molecules in the cytosol (i.e. the space
outside of all membrane-bound cytoplasmic organelles)
capable of binding to HA. One of them, receptor for
hyaluronan-mediated motility (RHAMM), probably exists in
both intra- and extracellular locations and affects cell locomotion (Turley 1992), controls entry into mitosis (Mohapatra
et al. 1996) and contributes to cell transformation (Hall et al.
1995). Another research group, while apparently dealing with
the same protein (named intracellular hyaluronan-binding
protein, IHABP) did not find it on the cell surface (Hofmann
et al. 1998) nor released into the intercellular space (Assmann
et al. 1998). In addition to RHAMM/IHABP, there is yet
another intracellular HA-binding protein, first isolated from rat
liver and also found on the surface of fibroblasts, named
HABP (Gupta et al. 1991). It is phosphorylated in the presence of HA (Ranganathan et al. 1994, 1995). Reduced levels
of HABP on sperm cell surface are associated with loss of
sperm motility (Ghosh et al. 2002), suggesting a crucial role
in fertilization. This HABP was later found to be identical
with P-32, a protein co-purified with the human premRNA
splicing factor-2 (SF2) (Deb and Datta 1996) and to have sequence homology with the complement protein gC1q-R (Das
et al. 1997).
Second, cytoplasmic and nuclear stainings have been
observed when a specific HA-binding probe is used for the localization of HA in cultured human aortic smooth muscle cells
and skin fibroblasts (Evanko and Wight 1999). Intracellular HA
staining resembles microtubule distribution (Evanko et al.
2004) and is proposed to be a component of nuclear matrix
and cytoskeleton, and to have a role in chromosome condensation (Evanko and Wight 1999). It has also been suggested
that hyperglycemic growth medium, commonly used in cell
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Microinjection-based probing of cytosolic hyaluronan
cultures, initiates cellular stress responses with intracellular synthesis of HA in mesangial cells (Ren et al. 2009; Wang and
Hascall 2009; Wang et al. 2011). In other studies, cytoskeletal
association of HA is consistent with RHAMM/IHABP interacting with microtubules and actin filaments (Assmann et al.
1999), and being a centrosomal protein important for the maintenance of the mitotic spindle and supporting its role in cell
division (Maxwell et al. 2003). The HABP/P32/gC1q has been
suggested to be a substrate for mitogen-activated protein
(MAP) kinase and capable of translocating into the nucleus
during cell division (Majumdar et al. 2002).
Third, HA is synthesized from the cytosolic pool of uridine
diphosphate (UDP)-N-acetylglucosamine (GlcNAc) and
UDP-glucuronic acid (GlcUA) by hyaluronan synthases
(HASs) (three isozymes called HAS1–3), located at plasma
membrane. While HAS enzymes themselves may form a pore
for the extrusion of the growing HA chain, another hypothesis
suggests that HA chain is first produced in the cytosol and
subsequently transported into the extracellular space by poreforming proteins of the multidrug resistance protein (MRP)
family (Ouskova et al. 2004; Prehm and Schumacher 2004).
Then, HA should be at least transiently present in the cytosol.
Evidence for this idea has been found in fibroblasts by
osmotic lysis into cytosol of endocytosis vesicles containing
HA oligosaccharides to compete with the export of endogenous HA by the multidrug-associated protein MRP5 (Schulz
et al. 2007).
In normal cellular conditions, latent HAS enzymes are
thought to stay in the endoplasmic reticulum (ER)-Golgi compartment and get inserted in the plasma membrane only when
active HA synthesis is possible (Mullegger et al. 2003; Rilla
et al. 2005). It has been proposed that premature activation of
HAS can take place under cellular stress, leading to intracellular synthesis of HA, and its extension out into the extracellular matrix as monocyte-binding cable structures (Hascall et al.
2004). This stress-related HA synthesis may also involve cytosolic exposure of HA.
In multiple myeloma, B-cells expressing a splice variant of
HAS1 were reported to produce intracellular, presumably
cytosolic HA which modulates RHAMM and leads to the
mitotic abnormalities in multiple myeloma (Adamia et al.
2005). In the osteosarcoma cell line MG-63, intracellular HA
produced by HAS2 accumulates in association with proliferation of the osteoblastic cells, although the source and exact
site of the cytoplasmic HA remained unclear (Nishida et al.
2005).
In contrast to the above reports suggesting the cytosolic
existence and functions for HA, there are also several reports
showing intracellular HA only in membrane-bound compartments in the endosome-lysosome pathway. Epidermal keratinocytes contain HA in cytoplasmic vesicles of various sizes,
often lining the internal face of the membranes of larger vesicles, while free cytosolic or nuclear HA is not found
(Pienimäki et al. 2001). In these cells, intracellular HA originates from cell surface or culture medium and has a half-life of
2–3 h (Tammi et al. 2001). Correspondingly, a large part of the
intracellular HA in aortic smooth muscle cells also colocalizes
with a lysosomal marker, suggesting that it is endocytosed and
destined for degradation, although some of the HA was found
in the nucleus (Evanko et al. 2004). In the same study it was
noticed that while endocytosed high-molecular-weight HA
remains in larger vesicles, HA of 50 or 300 kDa shows a
diffuse, network-like pattern, often in the perinuclear area.
As the exact role of intracellular HA has remained unclear
and the different cell lines may account for the discrepancies
in the earlier results, the present work was undertaken to
address the possible existence and function of cytosolic HA in
two different cell lines by microinjection, a technique not previously applied in this field. First, the fluorescent HA-binding
probe was injected into Has3-overexpressing MCF-7 cells and
nontransfected LP-9 cells to check whether HA is present in
specific sites of the cytosol accessible for binding by this
probe. Next, fluorescent HA and HA oligosaccharides were
injected into the cytosol to search for binding sites recognizing different HA chain lengths. For comparison, cells were
allowed to endocytose the same probes to investigate whether
they enter the cytosol. In addition, since there are contradicting hypotheses concerning the mechanism of HA translocation across the plasma membrane, we aimed at clarifying the
mechanism by injecting the cells with HA oligosaccharides,
glucose, mannose and HYAL to probe for their effects on the
synthesis of HA. Microinjections were chosen as a method,
because it allows relatively easy, fast and definite introduction
of substances directly into the cytosol of living cells without
harming the cells, and facilitates subsequent imaging of the
cells with confocal microscopy. The MCF-7 cells were chosen
for this study because their basic cell shape allows easy injections and when transfected with GFP-HAS3 they produce a
clearly visible HA coat, mainly dependent on the rate of HA
synthesis. The LP-9 cells were used as an example of a cell
type with spontaneously very active HA synthesis. Our results
showed no evidence for free cytosolic HA, nor perturbation
of HA synthesis by introducing HA oligosaccharides HA10
and HA14, or HYAL in the cytosol. However, microinjected
HA4 rapidly reduced and glucose increased HA synthesis as
measured by the size of the HA coat.
Results
Localization of endogenous intracellular HA
To explore the intracellular location of HA in MCF-7 and
LP-9 cells, we at first used the traditional protocol on other
cell types (Tammi et al. 2001), where cells fixed with aldehyde are first treated with HYAL to remove pericellular HA,
and then permeabilized to stain intracellular HA with HABC.
Using this method, intracellular HA was frequently found in
MCF-7 cells, and it was localized in vesicular structures of
different sizes, similar to what was found earlier in keratinocytes (Pienimäki et al. 2001; Tammi et al. 2001). The cytosol
and the nucleus were devoid of HA staining (Figure 1A
and B). Endogenous intracellular HA showed similar localization also in LP-9 cells (Figure 1C) and fibroblasts (data not
shown). The intracellular HA staining showed only occasional
co-localization with the hyaluronan synthase GFP-HAS3
(Figure 1B). The specificity of the HA staining was confirmed
by pretreatment of fHABC probe with HA10, which totally
abolished the signal (Figure 1E–G). The endogenous HA in
MCF-7 cells mostly co-localized with Alexa Fluor Hydrazide,
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Fig. 1. Localization of endogenous intracellular HA by fluorescent microscopy. Cultures of nontransfected (A and E), Has3-GFP-transfected MCF-7 cells (B and
F) or LP-9 mesothelioma cells (C and G) were fixed, pericellular HA removed with Streptomyces HYAL and cells permeabilized and stained for intracellular HA
with fHABC. In (E–G) the fHABC was pretreated with HA10 to check staining specificity (i.e. the HA10 competes for binding fHABC to cellular HA). Inset in
(B) shows magnification of the area pointed by the arrow in (B). To study hyaluronan endocytosis, in (D) live nontransfected MCF-7 cells were incubated with
0.5 mM Alexa Fluor 568 hydrazide for 2 h at 37°C and stained thereafter for endogenous HA with bHABC. (H and I) Magnifications of the areas pointed by the
arrows in (D). Red = HA; green = GFP-HAS3 or Alexa Fluor 568 hydrazide (D). Magnification bar 10 µm.
used as a marker of fluid-phase endocytosis (Figure 1D, H
and I), confirming the extracellular origin of the endogenous
intracellular HA in MCF-7 cells.
As HA in the cytosol may be lost during fixation and permeabilization, we next adopted a novel approach where we
microinjected fHABC into the cytosol of living cells. This
was assumed to bind to HA in the cytosolic side but be
excluded from the membrane-enclosed spaces like ER, Golgi
and endosomes. Two hours after microinjection, the injected
fHABC probe was found as a weak, diffuse signal in the
cytosol (Figure 2A and D) while in most of the injected
cells (80%) fHABC was also present as granular or vesicular structures of 0.2–2 µm in diameter (Figure 2A and D).
The microinjection marker containing a similar Alexa Fluor
conjugate as the injected fHABC showed an even distribution in the cytosol (data not shown) without similar accumulation in granular structures, indicating that the fluorescent
tag did not influence the localization of fHABC. The
fHABC signal was occasionally close to that of GFP-HAS3
(Figure 2D), suggesting that some of the granular staining
could be attached to HAS. However, this fHABC signal localization or intensity was not affected by HA10 or HYAL
(Figure 2B, C, E and F), indicating that the fHABC localization in these sites was not due to HA. LP-9, a mesothelioma
cell line spontaneously producing large quantities of HA,
showed microinjected fHABC pattern similar to that in
GFP-Has3-transfected
MCF-7
cells
(Figure
2G).
Occasionally, mitosis was observed in a microinjected cell,
but this caused no apparent change in the localization of
fHABC. To probe whether the intracellular location of
fHABC corresponded to the location of acidic compartments,
we added LysoTracker to the medium. However, there was
just occasional co-localization between the probe and this
lysosomal marker in 30% of MCF-7 and LP-9 cells
(Figure 2H and I). To check whether the microinjected
fHABC co-localized with endogenous HA, we stained the
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cells with bHABC for endogenous HA after microinjection
of fHABC (Figure 2J). Although fixation and staining
caused some dispersion of the granules formed by microinjected fHABC, we did not see any co-localization with endogenous HA visualized by traditional protocol (Figure 2J).
Finally, we also tested if the vesicular localization of microinjected fHABC was co-localized with microtubuli, possibly
associated with RHAMM (Evanko et al. 2004). MCF-7 cells
transfected with yellow fluorescent protein (YFP)-tagged
α-tubulin were injected with fHABC on the third day after
transfection as the microtubular structure was clearly visible.
There was no co-localization of the fHABC with the microtubuli (Figure 2K). In conclusion, the data indicate that the
majority of the fHABC enrichment sites were not associated
with lysosomes, Golgi, endosomes or intracellular HAS, nor
to endogenous HA or microtubuli.
Distribution of HA and its fragments microinjected into
cytosol
MCF-7 cells microinjected with fluorescent HA polymers and
its constituent mono- and oligosaccharides were examined
by confocal microscopy 1.5 and 6 h after the injection to investigate possible enrichment of HA binding sites in specific
cell domains. The localization of the injected fragments
was similar at both time points and did not show any
specific enrichment sites inside the cells. 2-aminoacridone
(AMAC)-labeled GlcUA was concentrated in speckled structures resembling small vesicles in the cytoplasm and it was
not found in the nucleus at all (Figure 3A). In contrast,
AMAC-labeled GlcNAc was distributed throughout the cell,
including the nucleus (Figure 3B).The injected HA4–HA28
oligosaccharides were evenly distributed in the cytosol, but
excluded from membrane-bound organelles like ER, Golgi
and mitochondria, seen as slightly darker areas in Figure 3C–
H; they were able to enter the nucleus (Figure 3C–H). However,
Microinjection-based probing of cytosolic hyaluronan
sizes of the fluorescently labeled oligosaccharides (HA4–
HA28) were detected in small vesicles, while no staining
outside the vesicles, either in the cytosol or in the nucleus,
was found even though the endosomal staining was strong
(Figure 3K–P). There was no difference in the number or
size of the HA-oligosaccharide positive vesicles between different oligosaccharide sizes (HA4–HA28). Thus, HA oligosaccharides originating from the extracellular space did not
reach cytosol, but remained in the endocytic, and probably
lysosomal compartments. The finding that AMAC-GlcNAc
but not AMAC-GlcUA was not restricted to endosomal structures was probably because of the stronger lipophilic character the AMAC-GlcNAc conjugate, allowing its penetration of
the membranes.
Fig. 2. Subcellular distribution of fHABC microinjected into cytosol
monitored by fluorescent microscopy. (A–C) fHABC microinjected in MCF-7
cells. (D–F) fHABC microinjected into Has3-GFP-transfected MCF-7 cells.
(B and E) the fHABC was pretreated with HA10 to block its HA binding
site. (C and F) fHABC was coinjected with Streptomyces HYAL (this
enzyme removes HA, if present). (G and H) LP-9 cells microinjected with
fHABC, (H) followed by incubation with Lysotracker® (green). (I)
Nontransfected MCF-7 cells were incubated with Lysotracker® after
microinjection of fHABC. (J) Nontransfected MCF-7 cells microinjected with
fHABC and then fixed and stained for endogenous HA. (K) Live
YFP-α-tubulin-transfected MCF-7 cells microinjected with fHABC.
Red = fHABC, green = Has3-GFP (D–F), Lysotracker® (H and I), HA (J) or
YFP-α-tubulin (K). Magnification bar 10 µm.
while HA4 and HA6 stained the nuclei evenly, oligosaccharides larger than HA8 were excluded from intranuclear structures resembling nucleoli (Figure 3E–H, arrows). The 25 kDa
HiLyte HA (HA120) was evenly distributed in the cytosol
and not in the membrane-bound organelles; it did not enter
the nucleus at all (Figure 3Q). It was not co-localized with
lysosomes (Figure 3R).
Intracellular localization of HA mono- and oligosaccharides
endocytosed from medium
By introducing labeled HA mono- and oligosaccharides in
the growth medium, the possibility of extracellullar HA fragments entering cytosol was studied in MCF-7 cells.
AMAC-labeled precursor sugars (GlcUA and GlcNAc) and
HA oligosaccharides of the range HA4–HA28 were incubated for 6 h and observed with confocal microscope.
Fluorescently labeled GlcUA remained within small intracellular vesicles (Figure 3I), while GlcNAc was distributed
throughout the cell, including the nucleus (Figure 3J). All
HA coat size in cells microinjected with HA
oligosaccharides, HYAL and glucose
Cells synthesizing large quantities of HA usually present a
thick cell surface coat of HA (Rilla et al. 2008, 2012), and
the thickness of this coat reflects the rate of HA synthesis
(Rilla et al. unpublished). The pericellular HA coat produced
by the MCF-7 cells is created mainly by active HA synthesis
(Kultti et al. 2006). The size of the coat was used here as an
estimate of HA synthesis in microinjected cells because this
sample number was too small to allow direct chemical quantification of the newly synthesized HA.
MCF-7 cells overexpressing GFP-Has3 were injected with
HA4–HA14 oligosaccharides to find out whether these fragments could compete with endogenous HA for binding to
the synthase, or plasma membrane pores for export, and
thereby inhibit HA synthesis (Prehm and Schumacher 2004).
Streptomyces HYAL injection was also used to check if the
growing HA chain could be reached from the intracellular
side. HA in the pericellular coat, visualized with fHABC at
4, 8 and 24 h after injection of the substances, was used as a
proxy for HA synthesis (Figures 4 and 5). HA oligosaccharides large enough (HA10) to compete for binding to HA
receptors (Tammi et al. 1998; Rilla et al. 2008) had no effect
on the size of the HA coat when injected into the cells
(Figure 5). Likewise, intracellular injection of 10 TRU/mL
HYAL (a concentration sufficient to rapidly remove extracellular HA when added in the medium) had no effect on the
size of HA coat (Figure 5). Instead, HA4 had a biphasic
effect on the coat size, with a transient 18% decline at 4 h
and a 30% increase at the 24 h time point (Figure 4D–F
and 5).
Glucose is a substrate for the synthesis of the HA precursors UDP-GlcNAc and UDP-GlcUA, and influences the synthesis of HA (Tammi et al. 2011). Therefore, it was expected
to serve as a positive control for using the coat size as a
measure of HA synthesis. Indeed, microinjection of glucose
was associated with increased pericellular HA coat at every
time point studied (Figures 4G–I and 5), probably by increasing the pools of intracellular precursor sugars. On the other
hand mannose, which is a structurally close isomer of
glucose, but metabolically more distant from the HA precursors, showed no significant effects on HA coat when injected
in the cells (Figures 4J–L and 5).
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Fig. 3. Subcellular distribution of fluorescently labeled HA oligosaccharides or polysaccharide, or constituent monosaccharides in MCF-7 cells. (A–H)
AMAC-labeled sugars (GlcUA and GlcNAc) or oligosaccharides (HA4—HA28) were microinjected into the cytosol and incubated for 1.5 h, while (I–P) the
same compounds were added in the culture medium and incubated with the cells for 6 h before imaging by confocal microscopy. (Q) Hilyte®-labeled HA
(HA120, red) was microinjected, while in HA120 was microinjected before incubation with Lysotracker® (lyso) (green). Arrows in (E–H) point to nuclear
substructures devoid of fluorescent oligosaccharides. Magnification bar 10 µm.
Discussion
Microinjections can be used to study specific intracellular processes in a limited number of single cells, but it is not the
most suitable method for studying dynamic processes in large
cell populations. When single cells are needed for quantitative
measurements, large numbers of microinjected cells are
required, but there is a limited time available for injections
and subsequent imaging. In the present study, intercellular differences in HA production and state of the cell cycle result in
variation that cannot be totally avoided. However, for intracellular visualization and subcellular localization of molecules
related to HA metabolism in qualitative terms, the method is
very convenient and helps to overcome the plasma membrane
barrier in the introduction of molecules into cells. Although it
is an artificial, potentially injurious method of introducing
substances into the cytosol, thorough optimization of the parameters minimizes the impact of the injection injury on results.
226
Earlier, Collis and colleagues microinjected HA of 60–600
kDa in 10T1/2 fibroblasts and noticed an increase in random
cell motility (Collis et al. 1998). They also incubated cells
with 700 kDa HA labeled with Texas Red and reported
rapid internalization and accumulation of HA in cellular
processes and nuclear area. The latter finding is in contrast
with the present data which indicate that endocytosed HA
regardless of size remains within endosome/lysosome type of
vesicles and that HA even when microinjected does not enter
the nucleus.
Our results on living cells suggest that there is a size-limit
between 5.6 and 25 kDa for the diffusion into the nucleus of
HA microinjected into cytosol. This can be compared with
the estimated 40–60 kDa barrier for diffusional entry of proteins into the nucleus. The highly hydrated and extended
structure explains why a considerably smaller molecular mass
limit was observed for HA.
Microinjection-based probing of cytosolic hyaluronan
Fig. 4. HA coat in cells microinjected with HA oligosaccharides, glucose and
mannose. Light microscopy was used to follow (4–24 h observations) the
effects of microinjection buffer (Co) (A–C), with HA tetrasaccharide (HA4)
(D–F), glucose (Glc) (G–I) or mannose (Man) (J–L) on HA coat of the
MCF-7 cells induced to express Has3-GFP. Magnification bar 10 µm.
Earlier, HA was reported to be found in nuclei when fixed
and permeabilized smooth muscle cells and fibroblasts were
stained with biotinylated HA-binding probe (Evanko and
Wight 1999). However, in the cell types used in the present
work HA was not seen in the nucleus of fixed cells. Also in
the experiments on living cells, when HA was introduced to
the cytosol, it failed to enter the nucleus. In some cases the
apparent inconsistency disappears if diffuse intracellular HA
signal is construed as coming from small vesicles rather than
cytosolic free intracellular HA (Evanko and Wight 1999).
Obviously, in addition to technical issues, different cell types,
their metabolic states and growth conditions are likely to influence the inconsistencies between our results and those of
previous papers. The widely used hyperglycemic media
induce intracellular HA synthesis in mesangial cells when
applied at a specific point of the cell cycle, an effect that also
requires cell division (Wang et al. 2011), but the applicability
of the findings made in mesangial cells for other cell types
awaits further studies. Since low-glucose (normoglycemic)
media were used throughout the present work, we do not
know whether a higher concentration of glucose in growth
medium would have affected the results.
After endocytosis, HA oligosaccharides remained in vesicles of a size distribution corresponding to that reported
earlier for HA in another cell type, keratinocytes (Tammi
et al. 2001). This suggests that endocytosed HA regardless of
size is mainly processed in the endosomal-lysosomal pathway
for catabolism also in MCF-7 and LP-9 cells.
Interestingly, the AMAC-labeled monosaccharide GlcNAc
showed a similar distribution whether microinjected or added
in the medium, suggesting a free mobility of this molecule
through cellular membranes, perhaps due to the increased
lipophilicity brought by AMAC. In contrast, AMAC-GlcUA,
Fig. 5. Integrated intensity of HA coat size in cells microinjected with HA oligosaccharides, mannose, glucose and Streptomyces HYAL. Has3-GFP expressing
cells were imaged 4, 8 and 24 h after microinjection of the indicated compounds, and the coat-associated fHABC fluorescence was quantitated. The results are
shown the as mean integrated intensity (area × intensity) of three separate experiments, 12–35 cells for each treatment in each experiment, and expressed as % of
control ± SE (n = 3). The differences between the groups compared with control were tested with univariate ANOVA, *P < 0.05, ***P < 0.001.
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H Siiskonen et al.
molecules with roughly the same size as the AMAC-GlcNAc
remained in vesicular structures after endocytosis and was
also concentrated in such structures after microinjection, and
did not enter the nucleus. The higher polarity of the molecule
could explain the inability of endocytosed AMAC-GlcUA to
escape membrane-bound organelles, but the reason for the
uptake of microinjected AMAC-GlcUA into similar structures
remains obscure.
Microinjected fHABC was generally localized throughout
the cytosol, but specifically concentrated in unidentified
granular or vesicular structures. However, fHABC accumulation in these structures was not because they would contain
HA, since blocking the HA-binding site of the probe by
HA10 and removal by HYAL of any HA that might be
present did not change the localization. In addition, the microinjected fHABC did not show any co-localization with the endogenous HA or with microtubuli. The localization of the
injected fHABC was also not affected by its fluorescent label
as the microinjection marker with a same Alexa Fluor tag was
evenly distributed in the cytosol. Since only a minor portion
of the injected HA-binding probe showed colocalization with
the lysosomal marker, the nature of the fHABC deposits
remains speculative. Some of this fHABC may represent
autophagosomes to be later fused with lysosomes. Autophagy
is an important function of mammalian cells to maintain
homeostasis and it is used for turnover of all cellular components (for review, see (Mijaljica et al. 2012)). In addition,
there may also be some nonspecific association with cytosolic
proteins, but the significance of these contacts remains irrelevant since the HABP does not normally exist in the cytosol.
It is widely accepted that mammalian HASs do not need a
primer to start HA synthesis, as only the UDP-precursor
sugars and Mg2+ or Mn2+ are required (Weigel and DeAngelis
2007). This is in line with the missing effects on coat size of
microinjected oligosaccharides HA10 and HA14, long enough
to displace HA from its receptors (Hardingham and Muir
1973; Tammi et al. 1998). Intriguingly, HA4, usually considered too short for competition with HA for receptors, first
reduced the coat at 4 h, but then increased the coat after 24 h.
The mechanisms behind these events remain open at the
moment, but HA4 can be speculated to have access to the
HA-binding site in the interior of the HAS enzymes to interfere with HA synthesis and to cause the initial decrease in
HA coat, while HA10 and HA14 could be too large for
similar effects. On the other hand, in 24 h the short HA4 may
be degradated to yield the monosaccharides for UDP-GlcUA
and UDP-GlcNAc synthesis to stimulate the synthesis of HA
(Vigetti et al. 2006; Kultti et al. 2009). The completion of
cytosolic degradation of HA10 and HA14 may be slower.
Intracellular injection of HYAL did not cause changes in the
HA coat, making it unlikely that HA would traverse in the
cytosol from HAS to a separate pore protein for delivery into
cell exterior. This supports the earlier findings indicating that
HAS is the only protein needed for the spatially coupled
events of HA synthesis and membrane translocation (Thomas
and Brown 2010; Hubbard et al. 2012; Medina et al. 2012).
In conclusion, our results suggest that HA is neither located
nor can naturally enter the cytosol or the nucleus under normal
physiological conditions. However, if HA fragments ≤ HA28
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(5.6 kDa) for some reason, such as under the circumstances
of ER stress (Hascall et al. 2004) have a chance to reach
the cytosol, they can also enter the nucleus, while HA120
(25 kDa) does not, findings consistent with the sizes of
HA in solution and the nuclear pore. Observations on HA coat
size as an estimate for the rate of HA synthesis support the
notion that before secretion, HA is not accessible to cytosolic
HA-binding proteins or HA oligosaccharides capable of competing for receptors, implying that HAS itself forms the pore
for HA translocation through plasma membrane. In contrast,
microinjection of HA4, an oligosaccharide too small to
compete with receptors, caused a transient reduction in HA
coat size, perhaps interfering with the synthesis/translocation
system of HAS.
Materials and methods
Cell culture
MCF-7 breast adenocarcinoma cells were cultured in
alphaMEM (Life Technologies, Paisley, Scotland; 5.56 mM
glucose) supplemented with 5% inactivated fetal bovine
serum (FBS) (HyClone, Logan, UT), 2 mM glutamine
(EuroClone, Milan, Italy), 50 µg/mL streptomycin sulfate and
50 U/mL penicillin (EuroClone).
Human mesothelial cells (LP-9) were cultured in MCBD
110 medium (Sigma, St. Louis, MO; 4 mM glucose) and 199
medium (Sigma; 5.56 mM glucose) in a 1:1 ratio, supplemented with 15% inactivated FBS, glutamine and antibiotics
as above, 10 ng/mL epidermal growth factor (EGF) (Sigma)
and 0.05 µg/mL hydrocortisone (Sigma). Cells were passaged
twice a week at a 1:10 split ratio using 0.05% trypsin
(w/v) 0.02% Ethylenediaminetetraacetic acid (EDTA) (w/v)
(Biochrom, Berlin, Germany).
Construction of MCF-7 cell line with doxycycline-inducible
overexpression of human HAS3 gene
EGFP-HAS3 (Rilla et al. 2012) was cut with NcoI and EcoRI
and inserted into penTTGmirc2 (Shin et al. 2006) and
then recombined with GatewayLR Clonase II enzyme mix
(Invitrogen, Paisley, UK) into pSLICK-hygromycin (Shin
et al. 2006) to make pLSICK-EGFP-HAS3-hyg. Lentiviruses
were produced as follows. HEK293T cells (3.5 × 106) were
seeded on a ø10 cm plate 1 day prior to transfection with four
plasmids: 6 µg of pSLICK-EGFP-HAS3-hyg and three plasmids encoding essential viral proteins (2.25 µg pMDlg/pRRE,
2.25 µg pRSV/REV and 1.5 µg pVSV). Plasmid DNA was
mixed with 500 µL OptiMEM (Invitrogen, Paisley, UK) and
40 µL of FugeneHD (Roche Diagnostics, Espoo, Finland).
The transfection mixture was added to the cells after a 20 min
incubation at room temperature. After 12–16 h, one volume of
DMEM containing 30% FBS was added to the cells. Viruses
were harvested after an additional 24 h incubation and titrated.
MCF-7 cells were plated on a ø 6 cm dish and transduced
with 6–9 × 104 viruses (GFP-HAS3) in the presence of polybrene. The next day the cells were switched to selection
medium containing 250 µg/mL hygromycin (Clontech
Laboratories, Mountain View, CA) for 3 to 4 days, and then
to maintenance medium containing 50 µg/mL hygromycin.
Following induction with 1 µg/mL doxycycline (Sigma),
Microinjection-based probing of cytosolic hyaluronan
hygromycin-resistant cells expressing GFP-HAS3 were sorted
with BD FACSAria III flow cytometer (BD, Franklin
Lakes, NJ).
Transfections
Semiconfluent MCF-7 cultures grown on gridded glass
bottom culture dishes (MatTek Corporation, Ashland, MA)
coated with collagen type I (BD Biosciences, Bedford, MA)
were transiently transfected with EGFP-HAS3 (Rilla et al.
2012) using ExGen 500 (Fermentas, Helsinki, Finland)
according to the manufacturer’s instructions and examined
one day after transfection. To examine the localization of
microinjected fluorescent HA binding complex in regard to
microtubuli, the MCF-7 cells were transiently transfected with
pEYFP-Tub Vector (Clontech Laboratories) encoding a fusion
protein of enhanced yellow fluorescent protein and human
α-tubulin as described above.
Fluorescent HA binding complex
The HA-binding probe consisting of cartilage link protein and
the G1 domain of aggrecan was purified from bovine articular
cartilage (Tammi et al. 1994) and labeled with a fluorescent
tag as described before (R.K. et al. 2008). In the present
experiments, Alexa Fluor 647 was used for HABC labeling
and Alexa Fluor 568 hydrazide as a microinjection marker
(both from Molecular Probes, Oregon, OR) to avoid spectral
overlap with GFP.
Staining of endogenous HA
To show the localization of endogenous HA in nontransfected
and transfected MCF-7 cells and nontransfected LP-9 cells,
semiconfluent cultures were grown on 8-well chambered
cover glass slides precoated with type I collagen. After
removal of the growth medium and wash with 0.1 M
Na-phosphate buffer (PB), pH 7.0, the cells were fixed with
4% paraformaledehyde for 1 h followed by washes in PB.
Extracellular HA was removed with 10 turbidity reducing
units/mL of Streptomyces HYAL (Seikagaku Kogyo Co.,
Tokyo, Japan) and then washed in PB and permeabilized with
0.1% Triton X-100, in PB and 1% BSA. Endogenous HA was
stained with fHABC, in 1% bovine serum albumin (BSA) in
PB overnight at 4°C. As a negative control, fHABC was preincubated with HA10 oligosaccharide in a 1:3 (w/w) ratio.
After staining, the chambers were washed in PB and imaged
with confocal microscopy. To confirm the localization of endogenous intracellular HA in endocytosed vesicles, the cells
were preincubated with 0.5 mM Alexa Fluor 568 hydrazide as
a fluid-phase endocytosis marker for 2 h, then washed in PB
and fixed with 4% paraformaldehyde-0.5% glutaraldehyde for
1 h followed by washes in PB. Extracellular HA was removed
with 5 TRU/mL Streptomyces HYAL (Seikagaku) and then
washed in PB, permeabilized with cold methanol and blocked
with 1% BSA to avoid nonspecific binding. Endogenous HA
was stained with biotinylated complex of HA -binding region
of bovine articular cartilage aggrecan G1 domain and link
protein (bHABC) (Tammi et al. 1994), diluted to 2.5 µg/mL
in 1% BSA, incubated overnight at 4°C and then washed in
PB and followed by incubation with fluorescein-conjugated
streptavidin (Vector Laboratories, Burlingame, CA) for 1 h as
a reporter. To check whether the microinjected fHABC localized with endogenous HA, the MCF-7 cells were first microinjected with fHABC as described below, incubated for 2 h at
37°C and then stained for endogenous HA with bHABC as
described above.
Fluorescent HA polymer probes
To monitor and track the microinjected HA molecules, the
polymers were labeled with a single fluorescent dye tag at the
reducing terminus; these methods minimize the disruption of
the HA structure and its interactions with other molecules.
For high efficiency labeling, two methods for tagging the HA
were used: (i) direct AMAC derivatization for oligosaccharides or (ii) enzymatic extension of a Hilyte 555-tagged HA4
into a 25 kDa polysaccharide.
AMAC-labeled HA fragments: HA oligosaccharides of 4–
14 monosaccharides units (HA4–14) were kindly donated by
Seikagaku while HA28 was prepared in-house (Lesley et al.
2000). For 2-AMAC labeling, the oligosaccharides (1 µmole),
D-glucuronic acid (GlcUA; Sigma), or N-acetylglucosamine
(GlcNAc; Sigma), were dried in 1.5 mL centrifuge tubes,
dissolved in 100 µL of 0.1 M AMAC (molecular probes) in
acetic acid/dimethyl sulphoxide (3/17, v/v), mixed with 100 µL
of freshly prepared 1 M cyanoborohydride (Sigma) in water,
and incubated overnight at 37°C. The reaction mixture was
applied on a 0.5 × 5 cm column of Superdex 30 (Pharmacia
GE) equilibrated and eluted with 12 mM NH4HCO3. The first
fluorescent peak, containing the labeled glycan, was collected
and dried in a centrifugal evaporator. The labeled products
were stored shielded from light at −70°C.
In vitro synthesis of fluorescent HA120 (25 kDa) polymers
The HA tetrasaccharide, HA4, and its amino-HA4 derivative
were prepared as described (Jing and DeAngelis 2004).
Amino-HA4 was derivatized with the N-hydroxysuccinimide
ester of Hilyte Fluor 555 (Anaspec) in 50% dimethyl sulfoxide and 100–200 mM sodium bicarbonate, pH 8.3. The derivatized HA4 was separated from free dye and amino-HA4 by
solid phase extraction (Strata C18; Phenominex, Torrance,
CA). The Hilyte 555-fluorescent HA4 molecules were then
extended to longer chains in reactions using 1.3 mg/mL HA
synthase PmHAS1–703 (as in (DeAngelis et al. 2003), but pretreated with Detoxi-Gel™ (Pierce) according to manufacturer
guidelines to remove endotoxin). The reaction was done in
8.5–10 mM UDP-GlcNAc, 8.5–10 mM UDP-GlcUA, 5 mM
MnCl2, 1 M ethylene glycol and 50 mM Tris, pH 7.2 at room
temperature for 60 h (Jing and DeAngelis 2004). To purify
the fluorescent HA, PmHAS was removed by chloroform
extraction and then UDP, a reaction byproduct, was removed
by extensive dialysis with the 3500 MWCO Slide-A-lyzer
dialysis cassette (Pierce). HA products were precipitated with
three volumes of ethanol and the pellets were redissolved in
water. The size of fluorescent HA polymer was analyzed on
agarose gels (0.7–1.2%; 1× TAE buffer = 40 mM Tris acetate,
2 mM EDTA) stained with Stains-All dye (0.005% w/v in
ethanol) compared with quasi-monodisperse HA standards
((DeAngelis et al. 2003), Hyalose, LLC, Oklahoma City).
The HA concentration was determined by the carbazole assay
229
H Siiskonen et al.
using a glucuronic acid standard (Bitter and Muir 1962). The
endotoxin level was determined by limulus amebocyte lysate
(LAL; Pierce) assay.
Microinjections
Semiconfluent cell cultures were microinjected with fHABC
(0.6 mg/mL), AMAC-labeled hyaluronan oligosaccharides
(1 µM), nonlabeled HA oligosaccharides (4 mg/mL HA4,
HA10 and HA14 (Seikagaku) or Streptomyces HYAL (10 turbidity reducing units/mL), dissolved in microinjection buffer
containing 48 mM K2HPO4, 4.5 mM KH2PO4 and 14 mM
NaH2PO4, pH 7.2. In addition, 10 mM AlexaFluor 568 hydrazide in 200 mM KCl was added to the microinjection buffer
in a 1:20 (v/v) ratio to help localization of the injected cells
afterwards. A further dilution of the injected solution in
a 1:10 ratio was perceived to occur after intracellular microinjection. Microinjections were performed at 37°C using
an Olympus IX-70 inverted microscope equipped with a
FemtoJet micromanipulator and an InjectMan NI2 microinjector (Eppendorf AG, Hamburg, Germany). Gridded glass
bottom culture dishes (MatTek Corporation, Ashland, MA)
were used to aid in the localization of the injected cells for
subsequent microscopic observation.
Optimization of microinjection conditions
A compensation pressure of 50 hPa was found to be sufficient
to prevent the capillary blockage and hydrostatic suction of
medium into the capillary. An injection pressure of 150 hPa
and an injection time of 0.5 s were chosen for all further experimentation, as preliminary experiments showed that using
these conditions cells experienced just a slight, transient
wave-like deformation, while increasing the pressure up to
300 hPa and the injection time to 1.5 s caused disintegration
of intracellular structures and the cells tended to detach from
substratum although the plasma membrane still remained
intact. The capillary was loaded with 3 µL of the solution to
be injected. Using a 40× objective injections were aimed at
the highest point of cytoplasm, but avoiding the nucleus. The
level of the capillary tip and the injection limits were customized in every experiment. About 15–30 cells could be
injected before the capillary showed signs of blockage or
became blunt. In all experiments, the cells were injected
within a 30–45 min period at 37°C.
Confocal microscopy
The fluorescent images were obtained with a 40× NA 1.3 oil
objective on a Zeiss Axio Observer inverted microscope
equipped with a Zeiss LSM 700 confocal module (Carl Zeiss
Microimaging GmbH, Jena, Germany). For live cell imaging,
a Zeiss XL-LSM S1 incubator with a temperature and CO2
control was utilized. ZEN 2009 software (Carl Zeiss
Microimaging GmbH) was used for the image processing.
The total intensity (mean intensity × area) of HA coat (Alexa
Fluor 647 nm) in each cell was quantified from 136 × 136 µm
areas with the ImageJ 1.45 s (National Institutes of Health).
For imaging, excitation at 488 nm was used for detection of
GFP-Has3, 555 nm for the microinjection marker (not shown
in figures) and 639 nm for localization of fHABC. In
230
experiments with AMAC-labeled oligosaccharides, we used
405 nm laser with beam splitters for the detection. Medium
was changed prior to microscopy. In HA coat analysis, a new
medium containing fHABC was exchanged for the spent
media 1 h prior to measurements.
Statistical analysis
The significances of differences between the groups in HA
coat measurements were tested using univariate analysis of
variance (ANOVA). Statistical tests were performed with
PASW Statistics 18 (IBM Corporation). Typically 12–35 cells
per experiment were averaged. Each experiment was performed three times.
Funding
This work was supported by the Academy of Finland to [M.T.];
Sigrid Juselius Foundation to [R.T.] and [M.T.]; Special
Government Funds (E.V.O.) for Kuopio University Hospital to
[M.T.]; Cancer Center of Eastern Finland to [R.T.] and [M.T.];
The Northern Savo Cultural Foundation to [H.S.]; The Northern
Savo Cancer Foundation to [H.S.]; The Emil Aaltonen
Foundation to [H.S.] and The Finnish Medical Foundation
to [H.S.].
Acknowledgements
Expert technical help from Tuula Venäläinen is gratefully
acknowledged.
Conflict of interest
None declared.
Abbreviations
AMAC, 2-aminoacridone; ANOVA, analysis of variance;
BSA, bovine serum albumin; EcoRI, Escherichia. coli restriction enzyme 1; EGF, epidermal growth factor; ER, endoplasmic reticulum; EDTA, ethylenediaminetetraacetic acid; FBS,
fetal bovine serum; fHABC, fluorescent HA binding complex;
GFP, green fluorescent protein; Glc, glucose; GlcNAc,
N-acetylglucosamine; GlcUA, glucuronic acid; HA, hyaluronan; HASs, hyaluronan synthases; IHABP, intracellular hyaluronan binding protein; LAL, limulus amebocyte lysate;
Man, mannose; MAP, mitogen-activated protein; MRP, multidrug resistance protein; PB, phosphate buffer; RHAMM, receptor for hyaluronan-mediated motility; SF2, splicing
factor-2; UDP, uridine diphosphate; YFP, yellow fluorescent
protein.
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