Growth, Differentiation and Sexuality

nearly all the label was present in water- and
alkali-soluble glucans. After a chase of radioactivity
and continued incubation in the presence
of unlabelled precursor, two patterns of
labelling were observed. Some of the hyphae
show displacement of the label to subapical regions,
indicating that these hyphae had grown
during the chase period. Other hyphae had
stopped growing, due to mechanical disturbancebythechaseprocedure,andretainedall
label in their apices. In both cases, however,
during the incubation in unlabelled medium
a considerable part of the label appeared in
the alkali-insoluble fraction, at the expense of
label in the water/alkali-soluble glucan fraction.
It was found that the glucan transferred
from the water- and alkali-soluble fractions
into the alkali-insoluble fraction almost exclusively
contained (1-3)-β-linkages.
– After pulse-labelling with [ 3 H]glucosamine
or [ 3 H]glucose, labelling patterns shown in
Fig. 4.1 were observed only after removal of
cytoplasm by extraction with ethanolic KOH.
Removal of the cytoplasm by mechanical
breakage of the hyphae resulted in disappearance
of labelled apices; labelled glucan was
solubilised, and labelled chitin was dispersed.
After a chase, however, all label, now present
subapically in growing hyphae and still apically
in non-growing ones, was resistant to
the shearing forces produced by mechanical
breakage. This indicates transfer of label
during the chase period from mechanically
fragile to rigid wall structures.
– Immediately after pulse-labelling with
[ 3 H]glucosamine, incubation of the alkaliinsoluble
wall residue with chitinase or hot
dilute mineral acid affected solubilisation of
most of the label incorporated into chitin. After
a chase of 60 min, the chitin became resistant
to such treatment. This may reflect the gap
between polymerisation and crystallisation of
chitin but also the protection of chitin chains
against chitinase by the attachment of glucan
chains.
– The growing hyphae, when extracted with alkali,
did not show microfibrils over their apices,
and apical chitin was rapidly disintegrated by
treatment with chitinase, again indicating the
absence of crystallinity. Chitin in non-growing
apices became resistant to chitinase, and did
reveal microfibrils after alkali extraction and
extraction of β-glucan with hot dilute acid.
Apical Wall Biogenesis 61
– By using glucose labelled with [ 3H] at either
the C3 or C2 position, and localising the label
by autoradiography before and after treatment
with periodate, it was found that the most apical
region of growing apices contained few (1-6)
linkages in the alkali-insoluble glucan. Subapically,
the number of (1-6) linkages in this glucan
rose rapidly. An abundance of (1-6) linkages
was also recorded in the alkali-insoluble glucan
which covered non-growing apices, so that
also in this respect the wall over these apices
became very similar to the subapical wall.
Importantly, in growing hyphae all the abovementioned
wall modifications continue beyond the extension
zone. We therefore surmise that, at the base
of the extension zone, these wall modifications have
only progressed sufficiently to produce a wall resisting
turgor but that the wall is not maximally
hardened. Although the most apical wall may be
protected by the structured cytoplasm, this may
explain why the wall bulges and eventually ruptures
just under the apex when turgor is suddenly
increased (see Sect. I).
B. Determinate Wall-Growth Model
for Budding Yeasts
Whereas in mycelial fungi the wall continuously
expands during growth, wall expansion in budding
and fission yeasts is discontinuous. Considering
only budding yeasts, after the bud has attained
a certain size it stops growing and then the
wall is apparently loosened again at predetermined
sites to allow for evaginations which grow into new
buds. There is a growing body of evidence indicating
that this process is similar to branching and
apical growth in mycelial fungi, with the important
difference that the gradient in wall synthesis is
less steep than that in hyphae, or becomes so during
bud growth (Staebell and Soll 1985; Klis et al.
2002).
Dimorphic fungi, such as C. albicans, areable
to modulate the pattern of wall deposition and
are thus able to switch between yeast and hyphal
growth, depending on environmental conditions
(see The Mycota, Vol. I, 1st edn., Chap. 8, and Vol.
VIII, Chap. 3). Careful measurements of wall expansion
in C. albicans (Soll et al. 1985; Staebell and
Soll 1985) have shown that the first phase of bud
growth is dominated by polarised wall expansion
whereas in the second phase, expansion of the bud
wall is more general. Only during the first phase