scholarly journals Rsr1 Palmitoylation and GTPase Activity Status Differentially Coordinate Nuclear, Septin, and Vacuole Dynamics in Candida albicans

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
T. Bedekovic ◽  
E. Agnew ◽  
A. C. Brand
Keyword(s):  
Candida Albicans ◽  
Mammalian Cells ◽  
Nuclear Division ◽  
Small Gtpase ◽  
Mitochondrial Morphology ◽  
Gtpase Activity ◽  
Polarized Growth ◽  
Directional Growth ◽  
Content Type ◽  
Septin Ring

ABSTRACT Directional growth and tissue invasion by hyphae of the pathogenic fungus, Candida albicans, are disrupted by deletion of the small GTPase, Rsr1, which localizes Cdc42 and its kinase, Cla4, to the site of polarized growth. We investigated additional abnormalities observed in rsr1Δ hyphae, including vacuole development, cytoplasm inheritance, mitochondrial morphology, septin ring organization, nuclear division and migration, and branching frequency, which together demonstrate a fundamental role for Rsr1 in cellular organization. Rsr1 contains a C-terminal CCAAX box, which putatively undergoes both reversible palmitoylation and farnesylation for entry into the secretory pathway. We expressed variants of Rsr1 with mutated C244 or C245, or which lacked GTPase activity (Rsr1K16N and Rsr1G12V), in the rsr1Δ background and compared the resulting phenotypes with those of mutants lacking Bud5 (Rsr1 GEF), Bud2 (Rsr1 GAP), or Cla4. Bud5 was required only for cell size and bud site selection in yeast, suggesting there are alternative activators for Rsr1 in hyphae. Septin ring and vacuole dynamics were restored by expression of unpalmitoylated Rsr1C244S, which localized to endomembranes, but not by cytoplasmic Rsr1C245A or GTP/GDP-locked Rsr1, suggesting Rsr1 functions at intracellular membranes in addition to the plasma membrane. Rsr1K16N or cytoplasmic Rsr1C245A restored normal nuclear division but not septin ring or vacuole dynamics. Rsr1-GDP therefore plays a specific role in suppressing START, which can be signaled from the cytosol. Via differential palmitoylation and activity states, Rsr1 operates at diverse cell sites to orchestrate proper nuclear division and inheritance during constitutive polarized growth. As cla4Δ phenocopied rsr1Δ, it is likely these functions involve Cdc42-Cla4 activity. IMPORTANCE Understanding how single eukaryotic cells self-organize to replicate and migrate is relevant to health and disease. In the fungal pathogen, Candida albicans, the small GTPase, Rsr1, guides the directional growth of hyphae that invade human tissue during life-threatening infections. Rsr1 is a Ras-like GTPase and a homolog of the conserved Rap1 subfamily, which directs migration in mammalian cells. Research into how this single GTPase delivers complex intracellular patterning is challenging established views of GTPase regulation, trafficking, and interaction. Here, we show that Rsr1 directly and indirectly coordinates the spatial and temporal development of key intracellular macrostructures, including septum formation and closure, vacuole dynamics, and nuclear division and segregation, as well as whole-cell morphology by determining branching patterns. Furthermore, we categorize these functions by differential Rsr1 localization and activity state and provide evidence to support the emerging view that the cytosolic pool of Ras-like GTPases is functionally active.

scholarly journals Heterogeneity in Mitochondrial Morphology and Membrane Potential Is Independent of the Nuclear Division Cycle in Multinucleate Fungal Cells

2012 ◽  
Vol 11 (3) ◽  
pp. 353-367 ◽  
Author(s):  
John P. Gerstenberger ◽  
Patricia Occhipinti ◽  
Amy S. Gladfelter
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
Mammalian Cells ◽  
Nuclear Division ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Morphology ◽  
Content Type ◽  
Expression Site ◽  
Zone Of Influence ◽  
Substantial Heterogeneity

ABSTRACT In the multinucleate filamentous fungus Ashbya gossypii , nuclei divide asynchronously in a common cytoplasm. We hypothesize that the division cycle machinery has a limited zone of influence in the cytoplasm to promote nuclear autonomy. Mitochondria in cultured mammalian cells undergo cell cycle-specific changes in morphology and membrane potential and therefore can serve as a reporter of the cell cycle state of the cytoplasm. To evaluate if the cell cycle state of nuclei in A. gossypii can influence the adjacent cytoplasm, we tested whether local mitochondrial morphology and membrane potential in A. gossypii are associated with the division state of a nearby nucleus. We found that mitochondria exhibit substantial heterogeneity in both morphology and membrane potential within a single multinucleated cell. Notably, differences in mitochondrial morphology or potential are not associated with a specific nuclear division state. Heterokaryon mutants with a mixture of nuclei with deletions of and wild type for the mitochondrial fusion/fission genes DNM1 and FZO1 exhibit altered mitochondrial morphology and severe growth and sporulation defects. This dominant effect suggests that the gene products may be required locally near their expression site rather than diffusing widely in the cell. Our results demonstrate that mitochondrial dynamics are essential in these large syncytial cells, yet morphology and membrane potential are independent of nuclear cycle state.

scholarly journals Clathrin- and Arp2/3-Independent Endocytosis in the Fungal Pathogen Candida albicans

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Elias Epp ◽  
Elena Nazarova ◽  
Hannah Regan ◽  
Lois M. Douglas ◽  
James B. Konopka ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Membrane Proteins ◽  
Fungal Pathogen ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Fungal Species ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Content Type ◽  
Actin Cables

ABSTRACT Clathrin-mediated endocytosis (CME) is conserved among eukaryotes and has been extensively analyzed at a molecular level. Here, we present an analysis of CME in the human fungal pathogen Candida albicans that shows the same modular structure as those in other fungi and mammalian cells. Intriguingly, C. albicans is perfectly viable in the absence of Arp2/3, an essential component of CME in other systems. In C. albicans, Arp2/3 function remains essential for CME as all 15 proteins tested that participate in CME, including clathrin, lose their characteristic dynamics observed in wild-type (WT) cells. However, since arp2/3 cells are still able to endocytose lipids and fluid-phase markers, but not the Ste2 and Mup1 plasma membrane proteins, there must be an alternate clathrin-independent pathway we term Arp2/3-independent endocytosis (AIE). Characterization of AIE shows that endocytosis in arp2 mutants relies on actin cables and other Arp2/3-independent actin structures, as inhibition of actin functions prevented cargo uptake in arp2/3 mutants. Transmission electron microscopy (TEM) showed that arp2/3 mutants still formed invaginating tubules, cell structures whose proper functions are believed to heavily rely on Arp2/3. Finally, Prk1 and Sjl2, two proteins involved in patch disassembly during CME, were not correctly localized to sites of endocytosis in arp2 mutants, implying a role of Arp2/3 in CME patch disassembly. Overall, C. albicans contains an alternative endocytic pathway (AIE) that relies on actin cable function to permit clathrin-independent endocytosis (CIE) and provides a system to further explore alternate endocytic routes that likely exist in fungal species. IMPORTANCE There is a well-established process of endocytosis that is generally used by eukaryotic cells termed clathrin-mediated endocytosis (CME). Although the details are somewhat different between lower and higher eukaryotes, CME appears to be the dominant endocytic process in all eukaryotes. While fungi such as Saccharomyces cerevisiae have proven excellent models for dissecting the molecular details of endocytosis, loss of CME is so detrimental that it has been difficult to study alternate pathways functioning in its absence. Although the fungal pathogen Candida albicans has a CME pathway that functions similarly to that of S. cerevisiae, inactivation of this pathway does not compromise growth of yeast-form C. albicans. In these cells, lipids and fluid-phase molecules are still endocytosed in an actin-dependent manner, but membrane proteins are not. Thus, C. albicans provides a powerful model for the analysis of CME-independent endocytosis in lower eukaryotes.

scholarly journals Rhb1 Regulates the Expression of Secreted Aspartic Protease 2 through the TOR Signaling Pathway in Candida albicans

2011 ◽  
Vol 11 (2) ◽  
pp. 168-182 ◽  
Author(s):  
Yu-Ting Chen ◽  
Chia-Ying Lin ◽  
Pei-Wen Tsai ◽  
Cheng-Yao Yang ◽  
Wen-Ping Hsieh ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Signaling Pathway ◽  
Virulence Factor ◽  
Nitrogen Availability ◽  
Target Genes ◽  
Small Gtpase ◽  
Aspartic Protease ◽  
Tor Signaling ◽  
Content Type

ABSTRACTCandida albicansis a major fungal pathogen in humans. InC. albicans, secreted aspartyl protease 2 (Sap2) is the most highly expressed secreted aspartic proteasein vitroand is a virulence factor. Recent research links the small GTPase Rhb1 toC. albicanstarget of rapamycin (TOR) signaling in response to nitrogen availability. The results of this study show that Rhb1 is related to cell growth through the control ofSAP2expression when protein is the major nitrogen source. This process involves various components of the TOR signaling pathway, including Tor1 kinase and its downstream effectors. TOR signaling not only controlsSAP2transcription but also affects Sap2 protein levels, possibly through general amino acid control. DNA microarray analysis identifies other target genes downstream of Rhb1 in addition toSAP2. These findings provide new insight into nutrients, Rhb1-TOR signaling, and expression ofC. albicansvirulence factor.

scholarly journals SR-Like RNA-Binding Protein Slr1 Affects Candida albicans Filamentation and Virulence

2013 ◽  
Vol 81 (4) ◽  
pp. 1267-1276 ◽  
Author(s):  
Chaiyaboot Ariyachet ◽  
Norma V. Solis ◽  
Yaoping Liu ◽  
Nemani V. Prasadarao ◽  
Scott G. Filler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Candida Albicans ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Umbilical Vein ◽  
Polarized Growth ◽  
Content Type ◽  
Fungal Burden

ABSTRACTCandida albicanscauses both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of bothSaccharomyces cerevisiaeandAspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence ofC. albicans. BLAST searches withS. cerevisiaeSR-like protein Npl3 (ScNpl3) identified twoC. albicansproteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, anotherSR-likeRNA-binding protein with no closeS. cerevisiaeortholog. Whereas ScNpl3 was critical for growth, deletion ofNPL3inC. albicansresulted in few phenotypic changes. In contrast, theslr1Δ/Δ mutant had a reduced growth ratein vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cellsin vitro. Mice infected intravenously with theslr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type orslr1Δ/Δ mutant complemented withSLR1(slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization ofslr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected withslr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cellsin vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin onslr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influencesC. albicansgrowth, filamentation, host cell interactions, and virulence.

scholarly journals Structures of the yeast dynamin-like GTPase Sey1p provide insight into homotypic ER fusion

2015 ◽  
Vol 210 (6) ◽  
pp. 961-972 ◽  
Author(s):  
Liming Yan ◽  
Sha Sun ◽  
Wei Wang ◽  
Juanming Shi ◽  
Xiaoyu Hu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Candida Albicans ◽  
Membrane Fusion ◽  
Mammalian Cells ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Helical Bundle ◽  
The Common ◽  
Insight Into ◽  
Common Function

Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases (GTPases), which include atlastin (ATL) in metazoans and Sey1p in yeast. In this paper, we determined the crystal structures of the cytosolic domain of Sey1p derived from Candida albicans. The structures reveal a stalk-like, helical bundle domain following the GTPase, which represents a previously unidentified configuration of the dynamin superfamily. This domain is significantly longer than that of ATL and critical for fusion. Sey1p forms a side-by-side dimer in complex with GMP-PNP or GDP/AlF4− but is monomeric with GDP. Surprisingly, Sey1p could mediate fusion without GTP hydrolysis, even though fusion was much more efficient with GTP. Sey1p was able to replace ATL in mammalian cells, and the punctate localization of Sey1p was dependent on its GTPase activity. Despite the common function of fusogenic GTPases, our results reveal unique features of Sey1p.

scholarly journals Rsr1 Focuses Cdc42 Activity at Hyphal Tips and Promotes Maintenance of Hyphal Development in Candida albicans

2012 ◽  
Vol 12 (4) ◽  
pp. 482-495 ◽  
Author(s):  
Rebecca Pulver ◽  
Timothy Heisel ◽  
Sara Gonia ◽  
Robert Robins ◽  
Jennifer Norton ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Rho Gtpase ◽  
Model Organism ◽  
Polarized Growth ◽  
Nucleotide Exchange ◽  
Transcriptional Program ◽  
Content Type ◽  
Hyphal Morphogenesis ◽  
Cell Shapes ◽  
Multiple Cell

ABSTRACTThe extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungusCandida albicansmake it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. InC. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeastSaccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth inC. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that inC. albicans, Rsr1 “fine-tunes” the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis.

scholarly journals The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection

2016 ◽  
Vol 84 (9) ◽  
pp. 2524-2533 ◽  
Author(s):  
Mary M. Weber ◽  
Robert Faris ◽  
Erin J. van Schaik ◽  
Juanita Thrasher McLachlan ◽  
William U. Wright ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Rho Gtpase ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Secretion System ◽  
Effector Proteins ◽  
Gtpase Activity ◽  
Content Type

Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial forC. burnetiiintracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment ofCoxiella burnetiiinfection and virulence in mammalian cell culture and mouse models of infection.

scholarly journals AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth inCandida albicans

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
H. C. Knafler ◽  
I. I. Smaczynska-de Rooij ◽  
L. A. Walker ◽  
K. K. Lee ◽  
N. A. R. Gow ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Cell Surface ◽  
Hyphal Growth ◽  
Specific Cell ◽  
Polarized Growth ◽  
Endocytic Recycling ◽  
Content Type ◽  
Wall Deposition ◽  
Cell Wall Deposition

ABSTRACTThe human fungal pathogenCandida albicansis known to require endocytosis to enable its adaptation to diverse niches and to maintain its highly polarized hyphal growth phase. While studies have identified changes in transcription leading to the synthesis and secretion of new proteins to facilitate hyphal growth, effective maintenance of hyphae also requires concomitant removal or relocalization of other cell surface molecules. The key molecules which must be removed from the cell surface, and the mechanisms behind this, have, however, remained elusive. In this study, we show that the AP-2 endocytic adaptor complex is required for the internalization of the major cell wall biosynthesis enzyme Chs3. We demonstrate that this interaction is mediated by the AP-2 mu subunit (Apm4) YXXΦ binding domain. We also show that in the absence of Chs3 recycling via AP-2, cells have abnormal cell wall composition, defective polarized cell wall deposition, and morphological defects. The study also highlights key distinctions between endocytic requirements of growth at yeast buds compared to that at hyphal tips and different requirements of AP-2 in maintaining the polarity of mannosylated proteins and ergosterol at hyphal tips. Together, our findings highlight the importance of correct cell wall deposition in cell shape maintenance and polarized growth and the key regulatory role of endocytic recycling via the AP-2 complex.IMPORTANCECandida albicansis a human commensal yeast that can cause significant morbidity and mortality in immunocompromised individuals. Within humans,C. albicanscan adopt different morphologies as yeast or filamentous hyphae and can occupy different niches with distinct temperatures, pHs, CO2levels, and nutrient availability. Both morphological switching and growth in different environments require cell surface remodelling, which involves both the addition of newly synthesized proteins as well as the removal of other proteins. In our study, we demonstrate the importance of an adaptor complex AP-2 in internalizing and recycling a specific cell surface enzyme to maintain effective polarized hyphal growth. Defects in formation of the complex or in its ability to interact directly with cargo inhibit enzyme uptake and lead to defective cell walls and aberrant hyphal morphology. Our data indicate that the AP-2 adaptor plays a central role in regulating cell surface composition inCandida.

scholarly journals Phosphoregulation of Nap1 Plays a Role in Septin Ring Dynamics and Morphogenesis in Candida albicans

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Zhen-Xing Huang ◽  
Pan Zhao ◽  
Gui-Sheng Zeng ◽  
Yan-Ming Wang ◽  
Ian Sudbery ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Mouse Model ◽  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Content Type ◽  
Septin Ring ◽  
Wide Range ◽  
Ring Dynamics ◽  
And Function

ABSTRACT Nap1 has long been identified as a potential septin regulator in yeasts. However, its function and regulation remain poorly defined. Here, we report functional characterization of Nap1 in the human-pathogenic fungus Candida albicans. We find that deletion of NAP1 causes constitutive filamentous growth and changes of septin dynamics. We present evidence that Nap1’s cellular localization and function are regulated by phosphorylation. Phos-tag gel electrophoresis revealed that Nap1 phosphorylation is cell cycle dependent, exhibiting the lowest level around the time of bud emergence. Mass spectrometry identified 10 phosphoserine and phosphothreonine residues in a cluster near the N terminus, and mutation of these residues affected Nap1’s localization to the septin ring and cellular function. Nap1 phosphorylation involves two septin ring-associated kinases, Cla4 and Gin4, and its dephosphorylation occurs at the septin ring in a manner dependent on the phosphatases PP2A and Cdc14. Furthermore, the nap1Δ/Δ mutant and alleles carrying mutations of the phosphorylation sites exhibited greatly reduced virulence in a mouse model of systemic candidiasis. Together, our findings not only provide new mechanistic insights into Nap1’s function and regulation but also suggest the potential to target Nap1 in future therapeutic design. IMPORTANCE Septins are conserved filament-forming GTPases involved in a wide range of cellular events, such as cytokinesis, exocytosis, and morphogenesis. In Candida albicans, the most prevalent human fungal pathogen, septin functions are indispensable for its virulence. However, the molecular mechanisms by which septin structures are regulated are poorly understood. In this study, we deleted NAP1, a gene encoding a putative septin regulator, in C. albicans and found that cells lacking NAP1 showed abnormalities in morphology, invasive growth, and septin ring dynamics. We identified a conserved N-terminal phosphorylation cluster on Nap1 and demonstrated that phosphorylation at these sites regulates Nap1 localization and function. Importantly, deletion of NAP1 or mutation in the N-terminal phosphorylation cluster strongly reduced the virulence of C. albicans in a mouse model of systemic infection. Thus, this study not only provides mechanistic insights into septin regulation but also suggests Nap1 as a potential antifungal target.

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