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internalized fluid-phase markers that non-specifically label all endo-
cytic vesicles, but preferentially endosomes on their way to lysosomes,
depending on the internalization conditions such as time and tem-
perature. Since fluid-phase markers (e.g. dextrans) do not bind to
cellular membranes, they are released into the cytoplasm when endo-
somes are lysed or pores are formed that are large enough to allow
passage of the markers. Internalized markers will be exposed to the
low pH environment of intact endosomes, whereas in the presence of
membrane-disrupting or pore-forming viruses, they will access the
pH neutral cytoplasm (Fig. 6A). Using fluid-phase markers of dif-
ferent molecular mass, release of the marker by endosome lysis can
be differentiated from release via a size-selective pore (Fig. 6A and B).
When a pH-sensitive (FITC) and a pH-insensitive (Cy5) derivative
of the same fluid-phase marker are internalized, flow cytometry of
intact cells can be used for quantification of marker uptake (reflected
by Cy5 fluorescence) and determination of the pH of the respective
compartment (reflected by the ratio of FITC and Cy5 fluorescence).
Detection of pH increase by this method indicates leakage of the
markers from acidic endosomes into the pH-neutral cytosol. The
number and pH of fluorescent endosomes can then be determined
by subjecting cell homogenates to single organelle flow analysis
(SOFA). Using fluid-phase markers of low and high molecular mass,
a differentiation can be made between virus-induced endosome rup-
ture and the formation of pores of a defined size appearing during
genome penetration. When adenovirus was internalized for 20 min,
this method revealed lysis of 40% of the endosomes that had been
labeled either with low or high molecular mass markers.
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Penetration of Major Group HRVs
Penetration of modified particles of the major group virus HRV14
into the cytoplasm occurs by rupturing the endosomal membrane
(Figs. 5 and 7). This is based on the following observations: HRV14
capsid proteins are detected in (isolated) endosomes under conditions
that block their conformational modification (20
C) and thus uncoat-
ing. No virus was present in isolated endosomes at 34
°
°
C, but 135S
and 80S particles were found in the cytosol.
84
Using our FACS and
