Proteomic profiling of the extracellular matrix (slime sheath) of <i>Dictyostelium discoideum</i>

Huber, Robert J.; O’Day, Danton H.

doi:10.1002/pmic.201500143

Cited by 17 publications

(16 citation statements)

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“…41,45 Recent proteomic profiling of conditioned media from developing cells, and the ECM surrounding the multicellular tissue commonly referred to as the slug, revealed the presence of a number of proteins linked to adhesion. 58,59 Our previous study characterized Cln3-deficiency phenotypes during the mid-to-late stages of Dictyostelium development. 29 Specifically, loss of Cln3 caused precocious development and enhanced slug migration.…”

Section: Discussionmentioning

confidence: 99%

“…54,57 Although CadA lacks a signal sequence for secretion, it has been detected in conditioned media from developing cells and as an integral component of the extracellular matrix (ECM) during the mid-to-late stages of Dictyostelium development. 58,59 Given these findings, and the observation that GFP-Cln3 localizes predominantly to the CV system in Dictyostelium (Fig. 2), 29 we also examined whether Cln3-deficiency had any effect on the secretion of CadA, and if defects in its secretion could explain the aberrant early development of cln3 ¡ cells.…”

Section: Cln3-deficiency Alters the Levels Of The Cell Adhesion Protementioning

confidence: 99%

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Aberrant adhesion impacts early development in aDictyosteliummodel for juvenile neuronal ceroid lipofuscinosis

Huber

Myre

Cotman

2016

Cell Adhesion & Migration

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Neuronal ceroid lipofuscinosis (NCL), also known as Batten disease, refers to a group of severe neurodegenerative disorders that primarily affect children. The most common subtype of the disease is caused by loss-of-function mutations in CLN3, which is conserved across model species from yeast to human. The precise function of the CLN3 protein is not known, which has made targeted therapy development challenging. In the social amoeba Dictyostelium discoideum, loss of Cln3 causes aberrant mid-to-late stage multicellular development. In this study, we show that Cln3-deficiency causes aberrant adhesion and aggregation during the early stages of Dictyostelium development. cln3 cells form ∼30% more multicellular aggregates that are comparatively smaller than those formed by wild-type cells. Loss of Cln3 delays aggregation, but has no significant effect on cell speed or cAMP-mediated chemotaxis. The aberrant aggregation of cln3 cells cannot be corrected by manually pulsing cells with cAMP. Moreover, there are no significant differences between wild-type and cln3 cells in the expression of genes linked to cAMP chemotaxis (e.g., adenylyl cyclase, acaA; the cAMP receptor, carA; cAMP phosphodiesterase, pdsA; g-protein α 9 subunit, gpaI). However, during this time in development, cln3 cells show reduced cell-substrate and cell-cell adhesion, which correlate with changes in the levels of the cell adhesion proteins CadA and CsaA. Specifically, loss of Cln3 decreases the intracellular level of CsaA and increases the amount of soluble CadA in conditioned media. Together, these results suggest that the aberrant aggregation of cln3 cells is due to reduced adhesion during the early stages of development. Revealing the molecular basis underlying this phenotype may provide fresh new insight into CLN3 function.

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Section: Discussionmentioning

confidence: 99%

Section: Cln3-deficiency Alters the Levels Of The Cell Adhesion Protementioning

confidence: 99%

Aberrant adhesion impacts early development in aDictyosteliummodel for juvenile neuronal ceroid lipofuscinosis

Huber

Myre

Cotman

2016

Cell Adhesion & Migration

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“…In addition, RgaA, PsaB, and CmbC were detected in NC4 ECM, while PsaA, CbpA (CBP1), and Ca 2+ ‐dependent cell‐to‐cell adhesion protein 3 (Cad3) were detected in WS380B ECM. While proteomics‐based analyses and western blotting support the secretion of CaM during multicellular development, and there is some evidence from immunolocalization experiments to support the presence of CaM in the ECM, mass spectroscopy did not detect CaM in the sheath ECM (Bakthavatsalam & Gomer, ; Suarez et al, ; O'Day et al, ; Huber & O'Day, ; Huber, ). These observations indicate that CaM might not reside long within the ECM, may be retained but undetectable due to its binding to CaMBPs, or may be present at levels that are too low for immunolocalization.…”

Section: Extracellular Matrix and Secreted Proteinsmentioning

confidence: 99%

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

et al. 2019

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This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin‐binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin‐dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin‐dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin‐dependent transdifferentiation to re‐establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin‐dependent germination of spores. Specific calmodulin‐binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.

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“…When conditions are sufficiently severe, the slug can form a fruiting body, where cells differentiate into a spore and stalk. During the formation of the slug and fruiting body, proteins and cellulose are deposited as an extracellular matrix, providing the organism with environmental protection and structural rigidity (1)(2)(3)(4). Cellulose is also found in the sheath that surrounds the cell aggregates and is deposited in the stalk, stalk cell walls, and spore coats (2,5).…”

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Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

Hobdey

Knott

Momeni

et al. 2016

Appl Environ Microbiol

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Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7A and DpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7A CBM and DpuCel7A CBM , which were recombinantly expressed in T. reesei. DdiCel7A CBM and DpuCel7A CBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The K i of cellobiose was significantly higher for DdiCel7A CBM and DpuCel7A CBM than for TreCel7A: 205, 130, and 29 M, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life. IMPORTANCE GH7CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for cellulose breakdown. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability. The present work demonstrates that two GH7 CBHs from social amoeba are surprisingly quite similar in structure and activity to the canonical GH7 CBH from the model biomass-degrading fungus T. reesei when tested under equivalent conditions (with added CBM-linker domains) on an industrially relevant substrate. Dictyostelia are a class of social amoebae (historically known as slime molds) containing four different groups of terrestrial bacterivores from the kingdom Amoebozoa. During vegetative growth, dictyostelia exist as single-celled organisms; upon starvation, a lack of nutrients becomes preventive for vegetative growth and the cells aggregate into a multicellular slug. Slugs have a defined posterior and anterior, have the ability to migrate, are sensitive to light and temperature, and exhibit an innate immune system. When conditions are sufficiently severe, the slug can form a fruiting body, w...

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Proteomic profiling of the extracellular matrix (slime sheath) of Dictyostelium discoideum

Cited by 17 publications

References 40 publications

Aberrant adhesion impacts early development in aDictyosteliummodel for juvenile neuronal ceroid lipofuscinosis

Aberrant adhesion impacts early development in aDictyosteliummodel for juvenile neuronal ceroid lipofuscinosis

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

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