Download Molecular Mechanisms of Exocytosis and Endocytosis

Independent Meeting held at the University of Edinburgh, March 23–25, 2003. Edited by D. Apps (Edinburgh). Supported by The Wellcome Trust,
The International Society for Neurochemistry, The Physiological Society, The Biochemical Society and The International Union of Biochemistry
and Molecular Biology.
Visualizing membrane trafficking using total
internal reflection fluorescence microscopy
V. Beaumont1
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.
Abstract
There is a dizzying array of fluorescent probes now commercially available to monitor cellular processes,
and advances in molecular biology have highlighted the ease with which proteins can now be labelled with
fluorophores without loss of functionality. This has led to an explosion in the popularity of fluorescence
microscopy techniques. One such specialized technique, total internal reflection fluorescence microscopy
(TIR-FM), is ideally suited to gaining insight into events occurring at, or close to, the plasma membrane of
live cells with excellent optical resolution. In the last few years, the application of TIR-FM to membrane
trafficking events in both non-excitable and excitable cells has been an area of notable expansion and
fruition. This review gives a brief overview of that literature, with emphasis on the study of the regulation
of exocytosis and endocytosis in excitable cells using TIR-FM. Finally, recent applications of TIR-FM to the
study of cellular processes at the molecular level are discussed briefly, providing promise that the future of
TIR-FM in cell biology will only get brighter.
Introduction to total internal reflection
fluorescence microscopy (TIR-FM)
TIR-FM is a technique that specifically illuminates fluorophores in a thin optical section above the interface between
two media with different diffractive indices. To achieve this,
laser light is directed obliquely at the interface between two
media from a high (n1 ) to a low (n2 ) diffractive index with
an incident angle greater than the critical angle (θ c ) of total
internal reflection (TIR). θ c is given by:
θc = sin−1 \(n2 /n1 )
(1)
Under these conditions, laser light is totally internally
reflected at the interface. Even so, some of the light still
penetrates the medium of lower diffractive index as an electromagnetic field called the ‘evanescent wave’. A key characteristic of the evanescent wave is that it propagates parallel
Key words: endocytosis, evanescent wave microscopy, exocytosis.
Abbreviations used: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor;
FRET, fluorescence resonance energy transfer; (e)GFP, (enhanced) green fluorescent protein;
PKC, protein kinase C; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein
receptor; TIR, total internal reflection; TIR-FM, total internal reflection fluorescence microscopy.
1
e-mail [email protected]
to the interface, vanishing exponentially with distance. The
decay length (d) of the evanescent wave along the depth of
field depends on the incident angle (θ), the wavelength of the
excitation beam (λ) and the diffractive indices of both media:
d = λ/[4π (n1 2 · sin2 \θ − n2 2 )]
(2)
The decay of the evanescent field with depth from the
interface z is described as:
I(z) = I0 e−z/d
(3)
Evanescent field penetration depths (the distance from the
interface whereby intensity decreases to I0 /e) of <100 nm
can be readily achieved from TIR at glass/aqueous interfaces,
providing an opportunity to selectively excite fluorophores
in an optical slice with the dimensions of thin electron
microscopy. This offers a 5–10-fold advantage over the optical
resolution achievable with confocal microscopy (≈500 nm).
If cells can be grown on, or temporarily adhered to,
glass coverslips, imaging of fluorophores at, or immediately
below, the plasma membrane can be achieved in live cells.
In pioneering work by Daniel Axelrod, this technique was
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first applied to the study of live cells, primarily to investigate
cell–substrate adhesions [1]. There are numerous excellent
reviews describing the TIR-FM technique in detail, ranging
from detailed explanations of the physics of evanescent field
generation [2–7] to practical implementation of TIR-FM, and
construction of either ‘through-the-prism’ or ‘through-theobjective’ TIR-F microscopes [8–10].
In ‘prism-type’ microscopes, incident light from a laser
strikes a prism that is coupled to the coverslip by glycerol
or another liquid of matching refractive index, and the light
emitted by fluorophores excited within the evanescent field is
collected by a high-numerical-aperture water immersion lens.
This technique allows fine control of the angle of incidence,
which is useful if one wishes to change the penetration
depth of the evanescent field. This can be achieved with high
precision by using acousto-optical detectors to rapidly alter
the incident angle of the laser light, and has been used to track
fluorophore motion into deeper regions of cells [11–13].
In through-the-objective TIR-FM, the incident laser light
is directed at an appropriate angle directly through the
objective of an inverted microscope. This imposes restraints
on the angle achievable from the incident laser light, and
hence on the resultant evanescent field depth. In practice,
incident angles exceeding θ c for a glass/water interface are
only achieved with objectives with numerical apertures of
>1.4, and d still cannot be controlled as accurately as in
prism-type TIR-FM. However, this small loss in precision
allows greater accessibility to the specimen – through-theobjective set-ups leave the top surface unobstructed, allowing
TIR-FM to be easily combined with other techniques. TIRFM has been used in combination with patch-clamping,
providing precise electrical control of individual cells, and
a synchrony between electrical and optical output [14–17].
Laser-trap forcemetry has also been combined with TIRFM in bovine chromaffin cells to investigate the directional
flow of an exocytic burst, as well as the differential force of
expulsion of secretory products [18].
Exocytosis and granule dynamics in
endocrine cells
Observing the motion and fusion of secretory granules
docked beneath the plasma membrane of isolated chromaffin
cells heralded the beginnings of using TIR-FM to explore the
molecular steps controlling exocytosis. By taking advantage
of the fact that certain fluorescent acidotropic dyes (such
as Acridine Orange) accumulate in acidic compartments of
cells, such as granules, it was possible to identify individual
fluorescing secretory granules within the evanescent field.
Detailed information about granule docking and mobility
was obtained by several groups [12,19–21], which showed
that granules above the plasma membrane are free to move,
albeit in a restricted manner, and can approach the membrane
in directed trajectories, where they subsequently become
relatively immobile (or docked). While the docking step was
reversible (granules could move away from the plasma mem
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brane after remaining resident for 10s of seconds), docked
granules were shown to fuse with the plasma membrane in
response to stimulation by a depolarizing solution, releasing
their contents along with a puff of fluorescent dye.
These studies opened up the possibility that the regulation
of pre-fusion steps could be studied in live cells in a noninvasive manner. In particular, the effects of cytoskeletal
modification using actin depolymerizers [22] and the effects
of neuromodulators on the regulation of granule mobility
were assessed directly. For instance, in bovine chromaffin
cells, Tsuboi et al. [23] showed that long-term (>30 min)
elevation of protein kinase C (PKC) activity approximately
doubled the morphologically docked pool of granules,
consistent with the earlier idea that PKC results in an increase
in the supply of granules to the plasma membrane, which
may explain its actions as a potent sectretagogue, despite its
concomitant inhibition of calcium influx through voltagegated calcium channels [24]. Interestingly, prior actin depolymerization of chromaffin cells rendered PKC activation
unable to increase the docked granule pool, suggesting that
PKC may influence granule docking by altering the cortical
actin network in these cells [25–27]. However, the fusion
machinery itself has also been proposed as a direct target in
the PKC-dependent enhancement of exocytosis [28–30].
In MIN6 and pancreatic β-cells, granules containing
insulin are released in response to elevated external glucose
concentrations. Using TIR-FM, recruitment of PKC to the
plasma membrane in response to glucose was shown by
constructing fusion constructs of enhanced green fluorescent
protein (eGFP) with PKC isoforms [31], suggesting that PKC
becomes well placed to regulate granule fusion, perhaps by
a direct effect on the fusion machinery. Other investigators
have shown a preferential release of the docked granule
pool containing insulin–GFP in MIN6 β-cells [32], and have
furthermore shown that this exocytosis could be inhibited
by introduction of the syntaxin H3 domain [to interfere
with SNARE complex formation, where SNARE is defined
as soluble N-ethylmaleimide-sensitive factor attachment
protein receptor], confirming that granule fusion (but not
pre-docking) is a SNARE-dependent process [33].
TIR-FM in PC-12 cells has also been used to study
organelle dynamics and the regulation of exocytosis. In cells
overexpressing a hybrid neuronal calcium sensor-1 protein (a
homologue of the non-mammalian protein frequenin) fused
to enhanced yellow fluorescent protein, synaptic-like microvescicles, but not granules, were shown to undergo an enhanced rate of both regulated and constitutive exocytosis [34].
Investigation of the coupling between exocytosis and
endocytosis has been assisted by the use of dual-colour
TIR-FM, whereby the proteins of interest, labelled with
different variants of GFPs, can be distinguished spectrally.
The emitted light from the two fluorophores is split by
the use of appropriate dichroics and filters, and each image
is illuminated at different areas on the chip of a CCD
camera [as achieved with the W-view optical system (A4314; Hamamatsu Photonics), the MultiSpec MicroImager
(Optical Insights) or the Cairn Optosplit]. This allows
Molecular Mechanisms of Exocytosis and Endocytosis
the simultaneous imaging of the spatio–temporal dynamics
of protein interactions. In PC-12 cells, synaptobrevin
(tagged with DsRed) and dynamin (tagged with eGFP)
have been imaged simultaneously. By capturing the exact
site and moment of exocytosis in response to electrical
stimulation (by watching the rapid and transient increase
in synaptobrevin–eGFP fluorescence), and comparing this
to the foci of endocytic activity, the authors showed that
clusters of DsRed–dynamin appeared randomly at the plasma
membrane following stimulation, gliding laterally prior to
disappearance (see also [35]). It was shown that more than
70% of the exocytic responses of synaptobrevin had no
immediate and specific dynamin counterpart at the same site.
Thus the stimulus-dependent recruitment of dynamin, and
subsequent ‘x–y’ movement, was interpreted as a ‘scanning’ of
the membrane for invaginated pits. This supports the notion
that endocytosis is not well coupled, at least temporally, to
regulated exocytosis in neuroendocrine cells [36].
On the same theme, another recent study, again in PC12 cells, utilized dual-colour TIR-FM to explore the fate
of post-exocytosed granules, and inferred a direct spatial
coupling of exocytosis with granule recapture (presumably
representing the endocytic event) [37]. Using the relatively
soluble granule peptide neuropeptide Y tagged with cyan
fluorescent protein (to detect the fusion of granules and
release of neuropeptide Y), in conjunction with either the
larger soluble granule protein tissue plasminogen activator
tagged with the yellow fluorescent protein Venus, or the
granule membrane protein phogrin tagged with DsRed,
the authors investigated the relationship between fusion
and post-exocytic membrane handling. They showed that,
following exocytosis, the membrane protein phogrin almost
entirely failed to disperse (surprisingly unlike the rapid
dispersal of synaptobrevin [36]), and that granules kept their
shape for several minutes following exocytosis, resealing and
re-acidifying at the same locus as the exocytic event itself.
The sequence of protein recruitment to the plasma
membrane controlling the endocytic phase of clathrindependent constitutive membrane retrieval has also been
scrutinized in fibroblasts [38]. Cells stably expressing GFPlabelled β-actin or dynamin-1–eGFP were transfected with a
clathrin-light-chain–DsRed fusion protein. The data showed
that, after remaining stationary for 10s of seconds at the
plasma membrane, clathrin-coated pits were internalized
immediately after a brief burst of dynamin recruitment, and
that movement of the clathin-coated structure away from
the membrane was accompanied by a transient assembly of
actin. Thus the authors suggest that dynamin may provide the
trigger, and actin the force, for movement of clathrin-coated
structures into the cytosol.
Exocytosis and vesicle dynamics in neurons
Real-time optical monitoring of synaptic vesicle recycling in
neurons became possible with the advent of the fluorescent
styryl dyes such as FM1-43 (for a review, see [39]). These
dyes partition into lipid membranes and become trapped
in recycled vesicles on endocytosis. Further stimulation of
synaptic transmission causes preloaded vesicles to release
their dye during subsequent rounds of exocytosis. Using
epifluorescence microscopy, this is observed as a loss of dye
from synaptic terminals.
The relative sparsity of TIR-FM studies in neurons
compared with neuroendocrine cells is related in part to the
difficulty in finding a suitable preparation to study – namely
synaptic terminals that are large, and which adhere closely
to glass when either acutely dissociated or cultured. These
criteria have been well met by the ON-type bipolar neurons
from the goldfish retina, whose giant synaptic terminals,
≈10 µm in diameter, are packed with synaptic vesicles, and
adhere well to glass in the absence of an associated postsynaptic cell following acute enzymic dissociation. With
the excellent background fluorescence rejection achievable
with TIR-FM, it became possible for the first time to image
single synaptic vesicle pre-fusion and fusion events at these
presynaptic sites [15]. ‘Docked’ vesicles (stationary for
>0.5 s) were apparent as punctate fluorescence superimposed
on a background of FM1-43 staining of the plasma membrane. The authors showed that there were also preferred
sites at which vesicles attached to the plasma membrane, and
furthermore that vesicles that were attached fused preferentially and rapidly on inititation of a depolarizing stimulus
(seen as a brightening and lateral spread of FM1-43), while
vesicles recruited to the plasma membrane contributed to
further exocytosis at later time points. In agreement with
studies on neuroendocrine cells, Zenisek et al. [15] were able
to determine that the docking, or ‘capture’, of vesicles at the
plasma membrane was indeed reversible. The time required
for docked vesicles to reach exocytic competence (the socalled priming step) was estimated as ≈210 ms.
In a later paper, the same authors also provided detailed
information about the nature of the exocytic event itself,
providing evidence that, on depolarization, FM1-43-stained
synaptic vesicles fuse completely enough with the plasma
membrane to allow the diffusion of dye into the surrounding
membrane in entirety. This finding was interpreted as
evidence of fusion occurring by the classical Heuser and Reese
view of exo-endocytosis – namely a flattening of the vesicle
with the plasma membrane on fusion, prior to endocytosis
[40], rather than by the opening of a transient fusion pore
(the ‘kiss-and-run’ exocytosis) first suggested by Meldolesi
and Ceccarelli [41].
Further work on goldfish bipolar neurons will no doubt
continue to provide exciting advances in our understanding
of the regulation and modulation of synaptic vesicle
recycling. However, long-term culture of these neurons, and
transfection with fluorescently tagged proteins, has proved
problematic, and may ultimately limit the usefulness of this
preparation in application of the molecular biology tools so
complimentary to the TIR-FM studies performed in other
cells. However, work in progress (V. Beaumont, unpublished
work) suggests that mouse bipolar neurons are equally well
suited to TIR-FM studies, and no doubt other preparations
will be discovered in time. Investigation of pre-fusion events
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and synaptic vesicle cycling, with the potential for singlemolecule resolution, in living mammalian neurons would
represent a unique functional assay for many of the synaptic
proteins already identified, and which can be altered using
transgenic technology.
From identifying single vesicles to single
molecular events
What does the future hold for cell biology using TIR-FM?
An exciting advance is the achievement of single-molecule
optical detection. The largest problem to overcome in singlemolecule fluorescence studies is typically the necessity to
reduce background noise in order to amplify the signal-tonoise ratio of single fluorophores. The most effective way to
reduce this background is to limit the excitation volume to the
region of interest. In 1995, Funatsu et al. [42] demonstrated
that the small excitation volume achievable with TIR-FM was
ideal for this purpose.
Fluorophores for single-molecule detection must be
relatively robust, having a large molar absorption coefficient
and a high quantum yield, and being relatively resistant
to photobleaching. Proteins conjugated to Alexa488, tetramethylrhodamine, Cy3, Alexa598 and Cy5 have successfully
met these criteria and have been used to visualize singlemolecule reactions in living cells. Binding of epidermal
growth factor (EGF) labelled in a 1:1 ratio with Cy3
to EGF receptors (EGFRs) and subsequent dimerization
of the EGF–EGFR complexes has been demonstrated at
the single-molecule level in live HeLa cells [43]. The
kinetics of dissociation between a chemoattractant and its
membrane receptor have also been studied using TIR-FM.
Dictoyostelium amoebae perform chemotaxis in response to
cAMP. To elucidate how cells sense gradients of cAMP, Ueda
et al. [44] synthesized Cy3-labelled cAMP and observed
its binding to individual receptors in cells undergoing
chemotactic migration.
In addition, proteins tagged with GFP and yellow fluorescent protein have also been imaged at the single-molecule
level. For instance, single GFP-labelled kinesin molecules
have been imaged travelling along Cy3-labelled microtubules
to determine how various microtubule-associated proteins
interfere with the attachment of these molecular motors [45].
The potential to detect the intermolecular interactions of
biomolecules at the single-molecule level using fluorescence
resonance energy transfer (FRET) combined with TIR-FM
has been elegantly demonstrated by Suzuki and co-workers
[46]. They devised a method whereby the fluorescence
spectrum of a single molecule can be assayed using TIR-FM
by passing the emitted light through a dispersion prism for
spectroscopy. Single-pair FRET was demonstrated between
Rhodamine Red (labelling the essential light chain of myosin)
and Cy5 (labelling the myosin heavy chain) in a reconstructed
double-labelled myosin motor domain, immobilized on a
quartz coverslip.
The structural rearrangement of a single protein has
also been detected. Tetramethylrhodamine was attached to
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specific cysteine residues in the voltage-sensing S4 segment
of Shaker K+ channels expressed in the plasma membrane of
oocytes [14]. Fluorescence intensity changed in response
to the voltage-sensitive movements of the protein channel
around the dye, as depolarization drove the S4 segment from
the resting to the activated conformation.
Conclusions
The compatibility of TIR-FM with other techniques, in
particular voltage clamping, means that it should be possible
to relate directly the structural rearrangements of individual
proteins, or the spatio–temporal co-ordination of protein
interactions, to physiological cell signalling events measured
simultaneously. Advances in molecular biology and protein
structure are rapidly uncovering the molecular players of
exocytosis and endocytosis. Clearly, one of the key objectives
in the future is to analyse comprehensively how these
molecular machines operate together to mediate the complex
and highly regulated process of synaptic vesicle fusion and
recycling, and TIR-FM techniques seem poised to do exactly
that. Furthermore, there are clear advantages of being able
to study these interactions ‘in situ’, where one does not
have to worry about the near-impossible task of reconstituting appropriate cellular conditions.
V.B. is supported financially by a postdoctoral fellowship from the
Medical Research Council, U.K.
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