scholarly journals Nek8445, a protein kinase required for microtubule regulation and cytokinesis in Giardia lamblia

2019 ◽  
Author(s):  
Kelly M. Hennessey ◽  
Germain C.M. Alas ◽  
Ilse Rogiers ◽  
Renyu Li ◽  
Ethan A. Merritt ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Giardia Lamblia ◽  
Cell Shape ◽  
Cell Imaging ◽  
Microtubule Organization ◽  
General Role ◽  
Ventral Disc ◽  
Altered Cell ◽  
Fixed Cell

AbstractGiardia has 198 Nek kinases whereas humans have only 11. Giardia has a complex microtubule cytoskeleton that includes eight flagella and several unique microtubule arrays that are utilized for parasite attachment and facilitation of rapid mitosis and cytokinesis. The need to regulate these structures may explain the parallel expansion of the number of Nek family kinases. Here we use live and fixed cell imaging to uncover the role of Nek8445 in regulating Giardia cell division. We demonstrate that Nek8445 localization is cell cycle regulated and this kinase has a role in regulating overall microtubule organization. Nek8445 depletion results in short flagella, aberrant ventral disc organization, loss of the funis, defective axoneme exit and altered cell shape. The axoneme exit defect is specific to the caudal axonemes, which exit from the posterior of the cell, and this defect correlates with rounding of the cell posterior and loss of the funis. Our findings implicate a role for the funis in establishing Giardia’s cell shape and guiding axoneme docking. On a broader scale our results support the emerging view that Nek family kinases have a general role in regulating microtubule organization.

scholarly journals Is the Secret of VDAC Isoforms in Their Gene Regulation? Characterization of Human VDAC Genes Expression Profile, Promoter Activity, and Transcriptional Regulators

2020 ◽  
Vol 21 (19) ◽  
pp. 7388
Author(s):  
Federica Zinghirino ◽  
Xena Giada Pappalardo ◽  
Angela Messina ◽  
Francesca Guarino ◽  
Vito De Pinto
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Expression Profiles ◽  
Transcriptional Regulators ◽  
General Role ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1

VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs’ presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon–intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1–3), their expression profiles, promoter activity, and potential transcriptional regulators. VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels, with a predominance of VDAC1 and VDAC2 over VDAC3. However, an RNA-seq cap analysis gene expression (CAGE) approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription factor binding sites (TFBSs) distribution in the promoters were investigated. The main regulators common to the three VDAC genes were identified as E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) transcription factor family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each VDAC gene isoform were identified, supporting the results in the literature, indicating a general role of VDAC1, as an actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of the VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoform gene.

scholarly journals Disc and Actin-Associated Protein 1 Influence Host Attachment in the Intestinal Parasite Giardia lamblia

2021 ◽  
Author(s):  
Melissa C. Steele-Ogus ◽  
Ava M. Obenaus ◽  
Nathan J. Sniadecki ◽  
Alexander R. Paredez
Keyword(s):  
Conformational Changes ◽  
Giardia Lamblia ◽  
Actin Polymerization ◽  
Intestinal Parasite ◽  
Interacting Proteins ◽  
Associated Proteins ◽  
Promising Area ◽  
Ventral Disc ◽  
And Function

The deep-branching eukaryote Giardia lamblia is an extracellular parasite that attaches to the host intestine via a microtubule-based structure called the ventral disc. Control of attachment is mediated in part by the movement of two regions of the ventral disc that either permit or exclude the passage of fluid under the disc. Several known disc-associated proteins (DAPs) contribute to disc structure and function, but no force-generating protein has been identified among them. We recently identified several Giardia actin (GlActin) interacting proteins at the ventral disc, which could potentially employ actin polymerization for force generation and disc conformational changes. One of these proteins, Disc and Actin Associated Protein 1 (DAAP1), is highly enriched at the two regions of the disc previously shown to be important for fluid flow during attachment. In this study, we investigate the role of both GlActin and DAAP1 in ventral disc morphology and function. We confirmed interaction between GlActin and DAAP1 through coimmunoprecipitation, and used immunofluorescence to localize both proteins throughout the cell cycle and during trophozoite attachment. Similar to other DAPs, the association of DAAP1 with the disc is stable, except during cell division when the disc disassembles. Depletion of GlActin by translation-blocking antisense morpholinos resulted in both impaired attachment and defects in the ventral disc, indicating that GlActin contributes to disc-mediated attachment. Depletion of DAAP1 through CRISPR interference resulted in intact discs but impaired attachment, gating, and flow under the disc. As attachment is essential for infection, elucidation of these and other molecular mediators is a promising area for development of new therapeutics against a ubiquitous parasite.

scholarly journals SMARCB1 Acts as a Quiescent Gatekeeper for Cell Cycle and Immune Response in Human Cells

2020 ◽  
Vol 21 (11) ◽  
pp. 3969 ◽  
Author(s):  
Sung Kyung Choi ◽  
Myoung Jun Kim ◽  
Jueng Soo You
Keyword(s):  
Cell Cycle ◽  
Immune Response ◽  
Cell Lines ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Fetal Lung ◽  
Human Cells ◽  
Lung Fibroblasts ◽  
General Role

Switch/sucrose non-fermentable (SWI/SNF)-related matrix-associated actin-dependent regulator of chromatin (SMARC) subfamily B member 1 (SMARCB1) is a core subunit of the switch/sucrose non-fermentable (SWI/SNF) complex, one of the adenosine triphosphate (ATP)-dependent chromatin remodeler complexes. The unique role of SMARCB1 has been reported in various cellular contexts. Here, we focused on the general role of the ubiquitous expression of SMARCB1 in a normal cell state. We selected ARPE19 (human primary retinal pigment epithelium) and IMR90 (from human fetal lung fibroblasts) cell lines as they have completely different contexts. Furthermore, although these cell lines have been immortalized, they are relatively close to normal human cells. The loss of SMARCB1 in ARPE19 and IMR90 cells reduced cell cycle progression via the upregulation of P21. Transcriptome analysis followed by SMARCB1 knockdown in both cell lines revealed that SMARCB1 was not only involved in cell maintenance but also conferred immunomodulation. Of note, SMARCB1 bound to interleukin (IL) 6 promoter in a steady state and dissociated in an active immune response state, suggesting that SMARCB1 was a direct repressor of IL6, which was further confirmed via loss- and gain-of-function studies. Taken together, we demonstrated that SMARCB1 is a critical gatekeeper molecule of the cell cycle and immune response.

scholarly journals Arabidopsis Class I Formin FH1 Relocates between Membrane Compartments during Root Cell Ontogeny and Associates with Plasmodesmata

2019 ◽  
Vol 60 (8) ◽  
pp. 1855-1870 ◽  
Author(s):  
Denisa Oulehlov� ◽  
Eva Koll�rov� ◽  
Petra Cifrov� ◽  
Přemysl Pejchar ◽  
Viktor Žï¿½rsk� ◽  
...  
Keyword(s):  
Cell Shape ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Cell Junctions ◽  
Class I ◽  
Microtubule Organization ◽  
Branch Number ◽  
General Role ◽  
Shape Complexity ◽  
Root Tissues

Abstract Formins are evolutionarily conserved eukaryotic proteins engaged in actin nucleation and other aspects of cytoskeletal organization. Angiosperms have two formin clades with multiple paralogs; typical plant Class I formins are integral membrane proteins that can anchor cytoskeletal structures to membranes. For the main Arabidopsis housekeeping Class I formin, FH1 (At3g25500), plasmalemma localization was documented in heterologous expression and overexpression studies. We previously showed that loss of FH1 function increases cotyledon epidermal pavement cell shape complexity via modification of actin and microtubule organization and dynamics. Here, we employ transgenic Arabidopsis expressing green fluorescent protein-tagged FH1 (FH1-GFP) from its native promoter to investigate in vivo behavior of this formin using advanced microscopy techniques. The fusion protein is functional, since its expression complements the fh1 loss-of-function mutant phenotype. Accidental overexpression of FH1-GFP results in a decrease in trichome branch number, while fh1 mutation has the opposite effect, indicating a general role of this formin in controlling cell shape complexity. Consistent with previous reports, FH1-GFP associates with membranes. However, the protein exhibits surprising actin- and secretory pathway-dependent dynamic localization and relocates between cellular endomembranes and the plasmalemma during cell division and differentiation in root tissues, with transient tonoplast localization at the transition/elongation zones border. FH1-GFP also accumulates in actin-rich regions of cortical cytoplasm and associates with plasmodesmata in both the cotyledon epidermis and root tissues. Together with previous reports from metazoan systems, this suggests that formins might have a shared (ancestral or convergent) role at cell–cell junctions.

scholarly journals A Genomic Multiprocess Survey of Machineries that Control and Link Cell Shape, Microtubule Organization, and Cell-Cycle Progression

2014 ◽  
Vol 31 (2) ◽  
pp. 227-239 ◽  
Author(s):  
Veronika Graml ◽  
Xenia Studera ◽  
Jonathan L.D. Lawson ◽  
Anatole Chessel ◽  
Marco Geymonat ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Shape ◽  
Cell Cycle Progression ◽  
Microtubule Organization ◽  
Cycle Progression

scholarly journals Nek8445, a protein kinase required for microtubule regulation and cytokinesis in Giardia lamblia

2020 ◽  
Vol 31 (15) ◽  
pp. 1611-1622
Author(s):  
Kelly M. Hennessey ◽  
Germain C. M. Alas ◽  
Ilse Rogiers ◽  
Renyu Li ◽  
Ethan A. Merritt ◽  
...  
Keyword(s):  
Cell Division ◽  
Protein Kinase ◽  
Giardia Lamblia ◽  
Cell Shape ◽  
Microtubule Array ◽  
Ventral Disk

Here we study the role of Nek8445 in regulating cell division and microtubule array organization in Giardia. Depletion of Nek8445 results in 87% of cells being stalled or blocked in cytokinesis. Nek8445 regulates ventral disk organization, funis formation, axoneme exit, and cell shape, all of which contribute to the observed cytokinesis defects.

scholarly journals Arabidopsis Class I Formin FH1 Relocates Between Membrane Compartments During Root Cell Ontogeny And Associates With Plasmodesmata

2018 ◽  
Author(s):  
Denisa Oulehlová ◽  
Eva Kollárová ◽  
Petra Cifrová ◽  
Přemysl Pejchar ◽  
Viktor Žárský ◽  
...  
Keyword(s):  
Cell Shape ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Cell Junctions ◽  
Class I ◽  
Microtubule Organization ◽  
Branch Number ◽  
General Role ◽  
Shape Complexity ◽  
Root Tissues

AbstractFormins are evolutionarily conserved eukaryotic proteins engaged in actin nucleation and other aspects of cytoskeletal organization. Angiosperms have two formin clades with multiple paralogs; typical plant Class I formins are integral membrane proteins that can anchor cytoskeletal structures to membranes. For the main Arabidopsis housekeeping Class I formin, FH1 (At3g25500), plasmalemma localization was documented in heterologous expression and overexpression studies. We previously showed that loss of FH1 function increases cotyledon epidermal pavement cell shape complexity via modification of actin and microtubule organization and dynamics. Here we employ transgenic Arabidopsis expressing green fluorescent protein-tagged FH1 (FH1-GFP) from its native promoter to investigate in vivo behaviour of this formin using advanced microscopy techniques. The fusion protein is functional, since its expression complements the fh1 loss-of-function mutant phenotype. Accidental overexpression of FH1-GFP results in a decrease in trichome branch number, while fh1 mutation has the opposite effect, indicating a general role of this formin in controlling cell shape complexity. Consistent with previous reports, FH1-GFP associates with membranes. However, the protein exhibits surprising actin- and secretory pathway-dependent dynamic localization and relocates between cellular endomembranes and the plasmalemma during cell division and differentiation in root tissues, with transient tonoplast localization at the transition/elongation zones border. FH1-GFP also accumulates in actin-rich regions of cortical cytoplasm and associates with plasmodesmata in both the cotyledon epidermis and root tissues. Together with previous reports from metazoan systems, this suggests that formins might have an ancestral role at cell-cell junctions.

scholarly journals Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast

2005 ◽  
Vol 16 (4) ◽  
pp. 1756-1768 ◽  
Author(s):  
Isabelle Loïodice ◽  
Jayme Staub ◽  
Thanuja Gangi Setty ◽  
Nam-Phuong T. Nguyen ◽  
Anne Paoletti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Microtubule Organization ◽  
Cellular Processes ◽  
Spindle Midzone ◽  
Fluorescence Live Cell Imaging

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Δ mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.

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