scholarly journals A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eun-Ah Park ◽  
Juri Kim ◽  
Mee Young Shin ◽  
Soon-Jung Park
Keyword(s):  
Cell Division ◽  
Giardia Lamblia ◽  
Specific Inhibitor ◽  
Site Directed Mutagenesis ◽  
Kinase Assay ◽  
Basal Bodies ◽  
Putative Open Reading Frame ◽  
Mitotic Spindles ◽  
Dividing Cells

Abstract Background Polo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation. Methods To assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites Results After incubating trophozoites with 5 μM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation. Conclusions These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.

scholarly journals A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

2020 ◽  
Author(s):  
Eun-Ah Park ◽  
Juri Kim ◽  
Mee Young Shin ◽  
Soon-Jung Park
Keyword(s):  
Cell Division ◽  
Mitotic Index ◽  
Giardia Lamblia ◽  
Specific Inhibitor ◽  
Subcellular Fractionation ◽  
Immunofluorescence Assay ◽  
Kinase Assay ◽  
Nuclear Fraction ◽  
Dividing Cells

Abstract Background Polo-like kinases (PLKs) are conserved serine/threonine kinase, regulating cell cycle. Giardia lamblia PLK (GlPLK) role in its cell has not been yet studied. Here, the function of GlPLK was investigated to provide the insight of roles in Giardia cell division, especially during cytokinesis and in flagella formation. Methods To access the function of GlPLK, Giardia trophozoites were treated with PLK-specific inhibitor, GW843286X (GW) or anti-glplk morpholino, then growth of the cells was monitored and phenotypic characteristics of GlPLK-inhibited cells were observed by using mitotic index and flow cytometry assay. Transgenic G. lamblia expressing GlPLK as a hemagglutinin (HA)-tagging was constructed and used for immunofluorescence assay to detect the localization of GlPLK, followed by the subcellular fractionation. Furthermore, kinase assay was performed to assess the phosphorylation activities of GlPLK by purified proteins or in vitro synthesized proteins. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites. Results After incubating trophozoites with 5 µM GW, the percentages of cells with four nuclei and/or longer flagella were increased. Immunofluorescence assays indicated that GlPLK was mainly localized at basal bodies and transiently localized at mitotic spindles in the dividing cells. Fractionation experiments demonstrated that GlPLK is present in the nuclear fraction, as did the centromeric histone H3. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells, i.e., increased mitotic index and flagella length. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis, and flagella. Conclusions These data indicated that GlPLK plays roles in Giardia cell division, especially during cytokinesis, and in flagella formation.

fumble Encodes a Pantothenate Kinase Homolog Required for Proper Mitosis and Meiosis in Drosophila melanogaster

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1267-1276
Author(s):  
Katayoun Afshar ◽  
Pierre Gönczy ◽  
Stephen DiNardo ◽  
Steven A Wasserman
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Cell Division Cycle ◽  
Protein Isoforms ◽  
Pantothenate Kinase ◽  
Male Germline ◽  
Dividing Cells ◽  
Mutant Cells ◽  
Mitosis And Meiosis

Abstract A number of fundamental processes comprise the cell division cycle, including spindle formation, chromosome segregation, and cytokinesis. Our current understanding of these processes has benefited from the isolation and analysis of mutants, with the meiotic divisions in the male germline of Drosophila being particularly well suited to the identification of the required genes. We show here that the fumble (fbl) gene is required for cell division in Drosophila. We find that dividing cells in fbl-deficient testes exhibit abnormalities in bipolar spindle organization, chromosome segregation, and contractile ring formation. Cytological analysis of larval neuroblasts from null mutants reveals a reduced mitotic index and the presence of polyploid cells. Molecular analysis demonstrates that fbl encodes three protein isoforms, all of which contain a domain with high similarity to the pantothenate kinases of A. nidulans and mouse. The largest Fumble isoform is dispersed in the cytoplasm during interphase, concentrates around the spindle at metaphase, and localizes to the spindle midbody at telophase. During early embryonic development, the protein localizes to areas of membrane deposition and/or rearrangement, such as the metaphase and cellularization furrows. Given the role of pantothenate kinase in production of Coenzyme A and in phospholipid biosynthesis, this pattern of localization is suggestive of a role for fbl in membrane synthesis. We propose that abnormalities in synthesis and redistribution of membranous structures during the cell division cycle underlie the cell division defects in fbl mutant cells.

Freeze-fracture analysis of membrane events during early neogenesis of cilia in Tetrahymena: changes in fairy-ring morphology and membrane topography

1983 ◽  
Vol 60 (1) ◽  
pp. 137-156
Author(s):  
L.A. Hufnagel
Keyword(s):  
Cell Division ◽  
Membrane Structure ◽  
Freeze Fracture ◽  
Fracture Analysis ◽  
Basal Bodies ◽  
Membrane Topography ◽  
Fairy Rings ◽  
Dividing Cells ◽  
Cortical System ◽  
Membrane Particle

A freeze-fracture analysis of early neogenesis of somatic and oral cilia of Tetrahymena was conducted using exponentially grown cultures and also cells induced to undergo oral reorganization. In this report, presumptive ciliary domains (PCDs), sites of future outgrowth of somatic cilia, are identified and their membrane structure is described in detail. The fairy ring, an array of membrane particles that occurs within the PCD and appears to be a precursor of the ciliary necklace, is described. A sequence of early stages in the formation of the ciliary necklace of somatic cilia is deduced from topographical information and membrane particle arrangements and numbers. Evidence is presented that basal bodies are seated at the cell surface prior to initiation of necklace assembly and a possible role for the basal body in necklace assembly is suggested. In dividing cells, new oral cilia grow out prior to orientation of cilia-parasomal sac complexes relative to cell axes. In dividing cells and during oral reorganization, new cilia also develop prior to their alignment into membranelles. Thus, growth of cilia is independent of their spatial orientation. Fairy rings were not observed during oral reorganization. During cell division, proliferation of new cilia is accompanied by the formation of a network of junctions between a cortical system of membranous cisternae, the cortical ‘alveoli’. These interalveolar junctions may serve as tracks for early positioning and orientation of new oral basal bodies.

Clinical, pharmacokinetic (PK), and pharmacodynamic findings from a phase I trial of an Eg5 inhibitor (AZD4877) in patients with refractory acute myeloid leukemia (AML)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 3580-3580
Author(s):  
G. Borthakur ◽  
S. Faderl ◽  
F. Ravandi ◽  
S. Padmanabhan ◽  
W. Stock ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Specific Inhibitor ◽  
Clinical Activity ◽  
Mitotic Spindles ◽  
Eligibility Criteria ◽  
Bone Marrow Biopsies ◽  
Dividing Cells ◽  
Tumor Studies ◽  
Refractory Acute Myeloid Leukemia ◽  
Kinesin Spindle Protein

3580 Background: AZD4877 is a potent, specific inhibitor of Eg5 (kinesin spindle protein). The only known function of Eg5 is to separate the centromeres during mitosis. Eg5 inhibition is thus specific for dividing cells, resulting in monoastral mitotic spindles (monoasters) and apoptotic cell death. Preclinically, hematologic tumor cell lines were generally more sensitive to AZD4877 than those derived from solid tumors. Methods: AZD4877 was administered IV daily x 3 as induction for up to 2 cycles, followed by consolidation (daily x 2) for up to 4 cycles. Eligibility criteria were standard. Results: Cohorts of 3–6 patients (pts) were treated at doses of 2, 4, 7, 10, 13, 16, and 18 mg/day. To date, 24 evaluable pts have received 33 induction and 3 consolidation cycles of treatment. Monoasters were detected at all dose levels evaluated (2, 7, 10, 13, and 18 mg/day). The T1/2 of AZD4877 ranged from 26 to 42 hr; PK were linear and drug levels non-cumulative. Myelosuppression, the dose limiting toxicity (DLT) in solid tumor studies, was not considered a DLT in this trial. Mucositis was the DLT at 18 mg/day; with 1 pt developing Gr 3 palmar-plantar syndrome at this dose. Bone marrow biopsies were undertaken at screening and as clinically indicated. In pts with evaluable biopsies, marrow blasts decreased by 60–80% in 2 pts, and by 30–50% in 3 pts, with no response in 3 pts. Conclusions: Enrollment is ongoing at 16 mg, the likely Phase 2 dose. Preliminary results suggest possible clinical activity in AML. An expansion phase at the MTD is planned. [Table: see text]

scholarly journals Cell division and the maintenance of epithelial order

2014 ◽  
Vol 207 (2) ◽  
pp. 181-188 ◽  
Author(s):  
Katerina Ragkousi ◽  
Matthew C. Gibson
Keyword(s):  
Cell Division ◽  
Dynamic Process ◽  
Rapid Growth ◽  
Single Cells ◽  
Building Blocks ◽  
Adherent Cells ◽  
Mitotic Spindles ◽  
Tissue Architecture ◽  
Tissue Organization ◽  
Dividing Cells

Epithelia are polarized layers of adherent cells that are the building blocks for organ and appendage structures throughout animals. To preserve tissue architecture and barrier function during both homeostasis and rapid growth, individual epithelial cells divide in a highly constrained manner. Building on decades of research focused on single cells, recent work is probing the mechanisms by which the dynamic process of mitosis is reconciled with the global maintenance of epithelial order during development. These studies reveal how symmetrically dividing cells both exploit and conform to tissue organization to orient their mitotic spindles during division and establish new adhesive junctions during cytokinesis.

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 Functional Analysis of the Cell Division Protein FtsW of Escherichia coli

2004 ◽  
Vol 186 (24) ◽  
pp. 8370-8379 ◽  
Author(s):  
Soumya Pastoret ◽  
Claudine Fraipont ◽  
Tanneke den Blaauwen ◽  
Benoît Wolf ◽  
Mirjam E. G. Aarsman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Site Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Intracellular Loop ◽  
Transmembrane Segments ◽  
Division Site ◽  
Amphiphilic Peptide ◽  
Cell Division Protein

ABSTRACT Site-directed mutagenesis experiments combined with fluorescence microscopy shed light on the role of Escherichia coli FtsW, a membrane protein belonging to the SEDS family that is involved in peptidoglycan assembly during cell elongation, division, and sporulation. This essential cell division protein has 10 transmembrane segments (TMSs). It is a late recruit to the division site and is required for subsequent recruitment of penicillin-binding protein 3 (PBP3) catalyzing peptide cross-linking. The results allow identification of several domains of the protein with distinct functions. The localization of PBP3 to the septum was found to be dependent on the periplasmic loop located between TMSs 9 and 10. The E240-A249 amphiphilic peptide in the periplasmic loop between TMSs 7 and 8 appears to be a key element in the functioning of FtsW in the septal peptidoglycan assembly machineries. The intracellular loop (containing the R166-F178 amphiphilic peptide) between TMSs 4 and 5 and Gly 311 in TMS 8 are important components of the amino acid sequence-folding information.

scholarly journals The Role of Capsid in HIV-1 Nuclear Entry

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1425
Author(s):  
Anabel Guedán ◽  
Eve R. Caroe ◽  
Genevieve C. R. Barr ◽  
Kate N. Bishop
Keyword(s):  
Nuclear Pore ◽  
Outer Face ◽  
Nuclear Entry ◽  
Nuclear Compartment ◽  
Critical Step ◽  
Technological Advances ◽  
Dividing Cells ◽  
Hiv 1 ◽  
Gain Access

HIV-1 can infect non-dividing cells. The nuclear envelope therefore represents a barrier that HIV-1 must traverse in order to gain access to the host cell chromatin for integration. Hence, nuclear entry is a critical step in the early stages of HIV-1 replication. Following membrane fusion, the viral capsid (CA) lattice, which forms the outer face of the retroviral core, makes numerous interactions with cellular proteins that orchestrate the progress of HIV-1 through the replication cycle. The ability of CA to interact with nuclear pore proteins and other host factors around the nuclear pore determines whether nuclear entry occurs. Uncoating, the process by which the CA lattice opens and/or disassembles, is another critical step that must occur prior to integration. Both early and delayed uncoating have detrimental effects on viral infectivity. How uncoating relates to nuclear entry is currently hotly debated. Recent technological advances have led to intense discussions about the timing, location, and requirements for uncoating and have prompted the field to consider alternative uncoating scenarios that presently focus on uncoating at the nuclear pore and within the nuclear compartment. This review describes recent advances in the study of HIV-1 nuclear entry, outlines the interactions of the retroviral CA protein, and discusses the challenges of investigating HIV-1 uncoating.

Sign in / Sign up

Export Citation Format

Share Document