Rosa Mouriño-Pérez | Centro de Investigación Científica y de Educación Superior de Ensenada, BC. (original) (raw)

Papers by Rosa Mouriño-Pérez

Fungal Biology, 2011

Keywords: Branching Cell wall Hyphal growth Septation Spitzenk€ orper a b s t r a c t Neurospora ... more Keywords: Branching Cell wall Hyphal growth Septation Spitzenk€ orper a b s t r a c t Neurospora crassa has been at the forefront of biological research from the early days of biochemical genetics to current progress being made in understanding gene and genetic network function. Here, we discuss recent developments in analysis of the fundamental form of fungal growth, development and proliferation e the hypha. Understanding the establishment and maintenance of polarity, hyphal elongation, septation, branching and differentiation are at the core of current research. The advances in the identification and functional dissection of regulatory as well as structural components of the hypha provide an expanding basis for elucidation of fundamental attributes of the fungal cell. The availability and continuous development of various molecular and microscopic tools, as utilized by an active and co-supportive research community, promises to yield additional important new discoveries on the biology of fungi.

PLoS Genetics, 2014

Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryo... more Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Dmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.

PLoS ONE, 2014

Septum formation in Neurospora crassa was studied by fluorescent tagging of actin, myosin, tropom... more Septum formation in Neurospora crassa was studied by fluorescent tagging of actin, myosin, tropomyosin, formin, fimbrin, BUD-4, and CHS-1. In chronological order, we recognized three septum development stages: 1) septal actomyosin tangle (SAT) assembly, 2) contractile actomyosin ring (CAR) formation, 3) CAR constriction together with plasma membrane ingrowth and cell wall construction. Septation began with the assembly of a conspicuous tangle of cortical actin cables (SAT) in the septation site .5 min before plasma membrane ingrowth. Tropomyosin and myosin were detected as components of the SAT from the outset. The SAT gradually condensed to form a proto-CAR that preceded CAR formation. During septum development, the contractile actomyosin ring remained associated with the advancing edge of the septum. Formin and BUD-4 were recruited during the transition from SAT to CAR and CHS-1 appeared two min before CAR constriction. Actin patches containing fimbrin were observed surrounding the ingrowing septum, an indication of endocytic activity. Although the trigger of SAT assembly remains unclear, the regularity of septation both in space and time gives us reason to believe that the initiation of the septation process is integrated with the mechanisms that control both the cell cycle and the overall growth of hyphae, despite the asynchronous nature of mitosis in N. crassa.

Fungal Genetics and …, Jan 1, 2006

By confocal microscopy, we analyzed microtubule (Mt) behavior during hyphal growth and branching ... more By confocal microscopy, we analyzed microtubule (Mt) behavior during hyphal growth and branching in a Neurospora crassa strain whose Mts had been tagged with GFP. Images were assembled spatially and temporally to better understand the 3-D organization of the microtubular cytoskeleton and a clearer view of its dynamics. Cytoplasmic Mts were mainly arranged longitudinally along the hyphal tube. Straight segments were rare; most Mts showed a distinct helical curvature with a long pitch and a tendency to intertwine with one another to form a loosely braided network throughout the cytoplasm. This study revealed that the microtubular cytoskeleton of a hypha advances as a unit, i.e., as the cell elongates, it moves forward by bulk flow. Nuclei appeared trapped in the microtubular network and were carried forward in unison as the hypha elongated. During branching, one or more cortical Mts became associated with the incipient branch and were pulled into the emergence of the branch. As extension of the branch and distortion of the Mts continued, Mts soon were severed with both new Mt ends (+ and À) present in the new branch. Although the exact mechanisms for addition Mt recruitment into the branch remains an open question, the recorded evidence indicates both bulk insertion of established cortical parent-hypha Mts as well as in situ polymerization were involved. The latter conclusion was supported by FRAP studies showing evidence of Mt nucleation and polymerization assembly in the growing tip of the developing branch. Nuclei entered the branch entrapped in the advancing network of Mts.

Fungal Genetics and …, Jan 1, 2008

Live-cell imaging methods were used to study microtubule dynamics in the apical regions of leadin... more Live-cell imaging methods were used to study microtubule dynamics in the apical regions of leading hyphae and germ tubes of Neurospora crassa expressing b-tubulin-GFP. Microtubule polymerization rates in hyphae of N. crassa were much faster than those previously reported in any other eukaryotic organism. In order to address the roles of motor proteins in microtubule dynamic instability in N. crassa, the microtubule-motor mutant strains, Dnkin and ro-1, were examined. Polymerization and depolymerization rates in leading hyphae of these strains were reduced by one half relative to the wild type. Furthermore, microtubules in germ tubes of wild type and microtubule-motor mutants exhibited similar dynamic characteristics as those in hyphae of mutant strains. Small microtubule fragments exhibiting anterograde and retrograde motility were present in leading hyphae of all strains and germ tubes of wild-type strains. Our data suggest that microtubule motors play important roles in regulating microtubule dynamic instability in leading hyphae but not in germ tubes.

Revista de biología …, Jan 1, 2006

Fungal Genetics and …, Jan 1, 2010

Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryote... more Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.

Revista de biología …, Jan 1, 2006

Methods in molecular …, Jan 1, 2010

The user has requested enhancement of the downloaded file. All in-text references underlined in b... more The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original docum and are linked to publications on ResearchGate, letting you access and read them immediately.

Fungal Biology, Jan 1, 2011

Keywords: Branching Cell wall Hyphal growth Septation Spitzenk€ orper a b s t r a c t Neurospora ... more Keywords: Branching Cell wall Hyphal growth Septation Spitzenk€ orper a b s t r a c t Neurospora crassa has been at the forefront of biological research from the early days of biochemical genetics to current progress being made in understanding gene and genetic network function. Here, we discuss recent developments in analysis of the fundamental form of fungal growth, development and proliferation e the hypha. Understanding the establishment and maintenance of polarity, hyphal elongation, septation, branching and differentiation are at the core of current research. The advances in the identification and functional dissection of regulatory as well as structural components of the hypha provide an expanding basis for elucidation of fundamental attributes of the fungal cell. The availability and continuous development of various molecular and microscopic tools, as utilized by an active and co-supportive research community, promises to yield additional important new discoveries on the biology of fungi.

Applied and …, Jan 1, 2003

Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its popula... more Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of V. cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with V. cholerae or the 0.6-m-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day ؊1 (4.2 day ؊1 ؎ 3.9) for V. cholerae and 0.1 to 9.7 day ؊1 (2.2 ؎ 2.8 day ؊1 ) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V. cholerae in seawater. Under the conditions tested, free-living V. cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10 4 cell ml ؊1 ) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V. cholerae in disease propagation and prevention during phytoplankton blooms.

Fungal Biology, 2011

Keywords: Branching Cell wall Hyphal growth Septation Spitzenk€ orper a b s t r a c t Neurospora ... more Keywords: Branching Cell wall Hyphal growth Septation Spitzenk€ orper a b s t r a c t Neurospora crassa has been at the forefront of biological research from the early days of biochemical genetics to current progress being made in understanding gene and genetic network function. Here, we discuss recent developments in analysis of the fundamental form of fungal growth, development and proliferation e the hypha. Understanding the establishment and maintenance of polarity, hyphal elongation, septation, branching and differentiation are at the core of current research. The advances in the identification and functional dissection of regulatory as well as structural components of the hypha provide an expanding basis for elucidation of fundamental attributes of the fungal cell. The availability and continuous development of various molecular and microscopic tools, as utilized by an active and co-supportive research community, promises to yield additional important new discoveries on the biology of fungi.

PLoS Genetics, 2014

Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryo... more Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Dmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.

PLoS ONE, 2014

Septum formation in Neurospora crassa was studied by fluorescent tagging of actin, myosin, tropom... more Septum formation in Neurospora crassa was studied by fluorescent tagging of actin, myosin, tropomyosin, formin, fimbrin, BUD-4, and CHS-1. In chronological order, we recognized three septum development stages: 1) septal actomyosin tangle (SAT) assembly, 2) contractile actomyosin ring (CAR) formation, 3) CAR constriction together with plasma membrane ingrowth and cell wall construction. Septation began with the assembly of a conspicuous tangle of cortical actin cables (SAT) in the septation site .5 min before plasma membrane ingrowth. Tropomyosin and myosin were detected as components of the SAT from the outset. The SAT gradually condensed to form a proto-CAR that preceded CAR formation. During septum development, the contractile actomyosin ring remained associated with the advancing edge of the septum. Formin and BUD-4 were recruited during the transition from SAT to CAR and CHS-1 appeared two min before CAR constriction. Actin patches containing fimbrin were observed surrounding the ingrowing septum, an indication of endocytic activity. Although the trigger of SAT assembly remains unclear, the regularity of septation both in space and time gives us reason to believe that the initiation of the septation process is integrated with the mechanisms that control both the cell cycle and the overall growth of hyphae, despite the asynchronous nature of mitosis in N. crassa.

Fungal Genetics and …, Jan 1, 2006

By confocal microscopy, we analyzed microtubule (Mt) behavior during hyphal growth and branching ... more By confocal microscopy, we analyzed microtubule (Mt) behavior during hyphal growth and branching in a Neurospora crassa strain whose Mts had been tagged with GFP. Images were assembled spatially and temporally to better understand the 3-D organization of the microtubular cytoskeleton and a clearer view of its dynamics. Cytoplasmic Mts were mainly arranged longitudinally along the hyphal tube. Straight segments were rare; most Mts showed a distinct helical curvature with a long pitch and a tendency to intertwine with one another to form a loosely braided network throughout the cytoplasm. This study revealed that the microtubular cytoskeleton of a hypha advances as a unit, i.e., as the cell elongates, it moves forward by bulk flow. Nuclei appeared trapped in the microtubular network and were carried forward in unison as the hypha elongated. During branching, one or more cortical Mts became associated with the incipient branch and were pulled into the emergence of the branch. As extension of the branch and distortion of the Mts continued, Mts soon were severed with both new Mt ends (+ and À) present in the new branch. Although the exact mechanisms for addition Mt recruitment into the branch remains an open question, the recorded evidence indicates both bulk insertion of established cortical parent-hypha Mts as well as in situ polymerization were involved. The latter conclusion was supported by FRAP studies showing evidence of Mt nucleation and polymerization assembly in the growing tip of the developing branch. Nuclei entered the branch entrapped in the advancing network of Mts.

Fungal Genetics and …, Jan 1, 2008

Live-cell imaging methods were used to study microtubule dynamics in the apical regions of leadin... more Live-cell imaging methods were used to study microtubule dynamics in the apical regions of leading hyphae and germ tubes of Neurospora crassa expressing b-tubulin-GFP. Microtubule polymerization rates in hyphae of N. crassa were much faster than those previously reported in any other eukaryotic organism. In order to address the roles of motor proteins in microtubule dynamic instability in N. crassa, the microtubule-motor mutant strains, Dnkin and ro-1, were examined. Polymerization and depolymerization rates in leading hyphae of these strains were reduced by one half relative to the wild type. Furthermore, microtubules in germ tubes of wild type and microtubule-motor mutants exhibited similar dynamic characteristics as those in hyphae of mutant strains. Small microtubule fragments exhibiting anterograde and retrograde motility were present in leading hyphae of all strains and germ tubes of wild-type strains. Our data suggest that microtubule motors play important roles in regulating microtubule dynamic instability in leading hyphae but not in germ tubes.

Revista de biología …, Jan 1, 2006

Fungal Genetics and …, Jan 1, 2010

Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryote... more Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.

Revista de biología …, Jan 1, 2006

Methods in molecular …, Jan 1, 2010

The user has requested enhancement of the downloaded file. All in-text references underlined in b... more The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original docum and are linked to publications on ResearchGate, letting you access and read them immediately.

Fungal Biology, Jan 1, 2011

Keywords: Branching Cell wall Hyphal growth Septation Spitzenk€ orper a b s t r a c t Neurospora ... more Keywords: Branching Cell wall Hyphal growth Septation Spitzenk€ orper a b s t r a c t Neurospora crassa has been at the forefront of biological research from the early days of biochemical genetics to current progress being made in understanding gene and genetic network function. Here, we discuss recent developments in analysis of the fundamental form of fungal growth, development and proliferation e the hypha. Understanding the establishment and maintenance of polarity, hyphal elongation, septation, branching and differentiation are at the core of current research. The advances in the identification and functional dissection of regulatory as well as structural components of the hypha provide an expanding basis for elucidation of fundamental attributes of the fungal cell. The availability and continuous development of various molecular and microscopic tools, as utilized by an active and co-supportive research community, promises to yield additional important new discoveries on the biology of fungi.

Applied and …, Jan 1, 2003

Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its popula... more Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of V. cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with V. cholerae or the 0.6-m-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day ؊1 (4.2 day ؊1 ؎ 3.9) for V. cholerae and 0.1 to 9.7 day ؊1 (2.2 ؎ 2.8 day ؊1 ) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V. cholerae in seawater. Under the conditions tested, free-living V. cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10 4 cell ml ؊1 ) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V. cholerae in disease propagation and prevention during phytoplankton blooms.