Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Cells of the yeast Saccharomyces cerevisiae are able to change their morphological
appearance in response to a variety of extracellular and intracellular signals. The
processes involved in morphogenesis are well characterised in this organism, but the
exact mechanism by which information emanating from the environment is integrated into
the regulation of the actin cytoskeleton and the yeast cell cycle, is still not clearly
understood. Considerable progress has, however, been made. The processes are
investigated on various levels including: (i) the nature of the signals required to elicit a
morphological adaptation, (ii) the mechanism by which these signals are perceived and
transmitted to the nucleus for gene transcription regulation (signal transduction pathways),
(iii) the role of the cytoskeleton, particularly actin, in morphogenesis, and (iv) the
relationship between cell cycle regulators and factors required for alterations in cellular
shape.
The focus of this study was on elements involved in the regulation of one of these
morphological processes, pseudohyphal formation, in S. cerevisiae. During pseudohyphal
differentiation normal oval yeast cells become elongated and mother and daughter cells
stay attached after cytokinesis to give rise to filaments. These filaments are able to
penetrate the growth substrate, a phenomenon referred to as invasive growth. Actin
remodelling is a prerequisite for the formation of elongated cells during pseudohyphal
development and invasive growth. Its main contribution to this event is the directing of
vesicles, containing cell wall constituents and enzymes, to specific sites of cell wall growth
at the cell periphery. In order to fulfil this cellular function, actin is regulated on several
levels. Signal transduction pathways that are activated in response to external nutritional
signals play important roles in the regulation of the actin cytoskeleton during pseudohyphal
differentiation. For this reason a literature review was compiled to introduce various
aspects of actin-structure, the regulation of this structure and the functions actin performs
during morphogenesis. The connection between signal transduction elements involved in
morphological processes and actin remodelling is also reviewed.
This study entailed the genetic analysis of numerous factors involved in the
regulation of pseudohyphal differentiation, invasive growth and starch metabolism. Several
transcriptional regulators playing a role in these phenomena were investigated. Apart from
the transcription factors, which include Mss11p, Msn1p, Ste12p, F108p,Phd1p and Tec1p,
additional elements ranging from transporters to G-proteins, were also investigated.
Mutant strains deleted for one or more of these factors were constructed and tested to
assess their abilities to form filaments that penetrate the growth substrate, and to utilise
starch as a carbon source. Complex genetic relationships were observed for various
combinations of these factors. Specifically, F108p,Msn1p and Ste12p were shown to act
independently in controlling invasive growth and starch metabolism, suggesting that these
factors are regulated by different signal transduction pathways. Mss11p, on the other hand, was found to play an indispensable role and seems to act as a downstream factor of
Msn1 p, Fl08p, Ste12p and Tec1 p. The exception to this is Phd1 p, since multiple copies of
PHD1 partially suppress the effect of a MSS11 deletion. The data suggests that Mss11 p
functions at the confluence of several signalling pathways controlling the transcriptional
regulation of genes required for invasive growth and starch degradation.
Different nutritional signals were also found to differentially regulate specific
signalling elements during the invasive growth response. For example, Tec1 p requires
Msn1 p activity in response to growth on media containing a limited nitrogen source. This
dependency, however, was absent when invasive growth was tested on glucose and
starch media.
Evidence was also obtained that confirmed the transcriptional co-regulation of
MUC1 and STA2. MUC1 encodes a mucin-like protein that is required for invasive growth
and pseudohyphal differentiation, whereas STA2 encodes a glucoamylase required for
starch degradation. Unpublished results indicated that several transcriptional regulators of
invasive growth also exert an effect on starch metabolism. The data generated during this
study complemented and confirmed published results. It also contributed to the
compilation of a more detailed model, integrating the numerous factors involved in these
signalling processes. / AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae gisselle beskik oor die vermoë om hul morfologiese voorkoms
in responstot 'n verskeidenheid van ekstrasellulêre en intrasellulêre seine te verander. Die
prosesse betrokke by morfogenese is goed gekarakteriseerd in hierdie organisme, maar
die presiese meganisme waardeur inligting vanuit die omgewing geïntegreer word in die
reguleringvan die aktien-sitoskelet en die gisselsiklus, word nog nie ten volle verstaan nie.
Aansienlike vordering in die verband is egter gemaak. Die prosesse word op verskeie
vlakke ondersoek, insluitende: (i) die aard van die seine wat benodig word om 'n
morfologiese aanpassing te inisïeer; (ii) die meganisme waardeur hierdie seine
waargeneem en herlei word na die selkern vir die regulering van geen-transkripsie
(seintransduksie paaie); (iii) die rol van die sitoskelet, spesifiek aktien, in morfogenese en
(iv) die verhouding tussen selsiklusreguleerders en faktore wat benodig word vir
verandering in selvorm.
Hierdie navorsing fokus op elemente betrokke by die regulering van een van hierdie
morfologiese prosesse in S. cerevisiae, naamlik pseudohife-vorming. Gedurende
pseudohife-differensiëring neem tipiese ovaalvormige selle 'n verlengde voorkoms aan wat
tot die vorming van filamente lei. Hierdie filamente is in staat om die groeisubstraat te
penetreer, 'n verskynsel bekend as penetrasie-groei. Aktienherrangskikking is 'n
voorvereiste vir die vorming van verlengde selle tydens pseudohife-ontwikkeling. Die
hoofbydrae van aktien tot hierdie verskynsel is die oriëntering van uitskeidingsvesikels,
wat selwandkomponente en ensieme bevat, na spesifieke areas van selwandgroei op die
seloppervlak. Aktien word op verskeie vlakke gereguleer om hierdie sellulêre funksie te
vervul. Seintransduksiepaaie wat geaktiveer word in respons tot ekstrasellulêre
voedingsseine speel 'n belangrike rol in die regulering van die aktien-sitoskelet tydens
pseudohife-differensiëring. Op grond hiervan is 'n literatuuroorsig saamgestel vir die
bekendstelling van verskeie aspekte van aktienstruktuur, die regulering van hierdie
strukture en die funksies wat deur aktien gedurende morfogenese vervul word. Die
verband tussen seintransduksie-elemente betrokke by morfologiese prosesse en aktien
herrangskikkingword ook behandel.
Hierdie studie het die genetiese analisering van verskeie faktore betrokke by
pseudohife-differensiëring, penetrasie-groei en styselmetabolisme, behels. Verskeie
transkripsionele reguleerders wat In rol speel in hierdie prosesse was bestudeer. Buiten
die transkripsiefaktore Mss11p, Msn1p, Ste12p, F108p,Phd1P en Tec1p, was addisionele
faktore, wat gewissel het van transporters tot G-proteïene, ook ondersoek. Mutante-rasse
met geendelesies vir een of meer van hierdie faktore is gekonstrueer en getoets om vas te
stel hoe dit hul vermoë raak om penetrerende filamente te vorm, asook om te bepaal of
stysel as koolstofbron gebruik kan word. Komplekse genetiese interaksies vir verskeie
kombinasies van hierdie faktore is waargeneem. Dit was waargeneem dat F108p,Msn1p en Ste12p onafhanklik funksioneer tydens die regulering van penetrasie-groei en
styselmetabolisme, wat impliseer dat hierdie faktore deur verskillende
seintransduksiepaaie gereguleer word. Mss11 p word beskou as In onmisbare rolspeler in
hierdie prosesse en dit kom voor asof hierdie protein as 'n stroom-af faktor is en vereis
word vir die funksionering van Msn1p, F108p, Ste12p en Tec1p. Phd1p is egter 'n
uitsondering, aangesien veelvuldige kopieë van PHD1 die effek van 'n MSS11-delesie
gedeeltelik oorkom. Die data impliseer dat Mss11 p by die samevloei van verskeie
seintransduksiepaaie, benodig vir die transkripsionele regulering van gene betrokke by
penetrasie-groei en styselmetabolisme, funksioneer.
Dit was ook waargeneem dat verskillende voedingsseine die faktore betrokke by die
penetrasie-groeirespons differensieel reguleer. Tec1 p byvoorbeeld benodig Msn1paktiwitieit
in respons tot groei op media met 'n beperkte stikstofbron. Hierdie afhanklike
interaksie is egter afwesig wanneer penetrasie-groei bestudeer word op glukose- en
styselmedia.
Resultate wat die gesamentlike transkripsionele regulering van MUC1 en STA2
bevestig, is ook verkry. MUC1 kodeer vir 'n mukienagtige proteïen wat benodig word vir
pseudohife-vorming en penetrasie-groei, terwyl STA2 kodeer vir 'n glukoamilase
essensieël vir styselafbraak. Ongepubliseerde resultate dui daarop dat verskeie
transkripsionele reguleerders van penetrasie-groei ook In effek uitoefen op
styselmetabolisme. Die data wat gegenereer is tydens hierdie studie komplementeer en
bevestig reeds gepubliseerde resultate. Dit het ook bygedra tot die samestelling van 'n
gedetaileerde model wat die verskillende faktore, betrokke by hierdie
seintransduksieprosesse, integreer.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/49972 |
| Date | 03 1900 |
| Creators | Van Dyk, Dewald, 1975- |
| Contributors | Pretorius, I. S., Bauer, F. F., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 146 p. : ill. |
| Rights | Stellenbosch University |
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