Live Cell Imaging of the Schizosaccharomyces pombe Sexual Life Cycle

2017 ◽  
Vol 2017 (10) ◽  
pp. pdb.prot090225 ◽  
Author(s):  
Laura Merlini ◽  
Aleksandar Vjestica ◽  
Omaya Dudin ◽  
Felipe Bendezú ◽  
Sophie G. Martin
Keyword(s):  
Life Cycle ◽  
Schizosaccharomyces Pombe ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Sexual Life ◽  
Sexual Life Cycle

scholarly journals Live cell imaging of the HIV-1 life cycle

2008 ◽  
Vol 16 (12) ◽  
pp. 580-587 ◽  
Author(s):  
Edward M. Campbell ◽  
Thomas J. Hope
Keyword(s):  
Life Cycle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Hiv 1

scholarly journals Adaptor protein Bbc1 regulates localization of Wsp1 and Vrp1 during endocytic actin patch assembly

2018 ◽  
Author(s):  
Cameron MacQuarrie ◽  
MariaSanta Mangione ◽  
Robert Carroll ◽  
Michael James ◽  
Kathleen L. Gould ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Schizosaccharomyces Pombe ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Adaptor Protein ◽  
Live Cell ◽  
Endocytic Vesicle ◽  
Actin Networks ◽  
Binding Partner ◽  
Actin Patch

ABSTRACTArp2/3 complex-nucleated branched actin networks provide the force necessary for endocytosis. The Arp2/3 complex is activated by Nucleation Promoting Factors (NPFs) including the Schizosaccharomyces pombe proteins WASp Wsp1 and myosin-1 Myo1. There are >40 known yeast endocytic proteins with distinct spatial and temporal localizations and functions; however, it is still unclear how these proteins work together to drive endocytosis. We used quantitative live cell imaging to determine the function of the uncharacterized S. pombe protein Bbc1. We discovered Myo1 interacts with and recruits Bbc1 to sites of endocytosis. Bbc1 competes with verprolin Vrp1 for Myo1 binding, thus releasing Vrp1 and its binding partner Wsp1 from Myo1. Normally Myo1 remains at the base of the endocytic invagination and Vrp1-Wsp1 internalize with the endocytic vesicle; however, in the absence of Bbc1, a portion of Vrp1-Wsp1 remains with Myo1 at the base of the invagination and endocytic invaginations are twice as long. We propose that Bbc1 disrupts a transient Myo1-Vrp1-Wsp1 interaction and limits Arp2/3 complex-nucleation of actin branches at the plasma membrane.

scholarly journals Kinesin-8B controls basal body function and flagellum formation and is key to malaria parasite transmission

2019 ◽  
Author(s):  
Mohammad Zeeshan ◽  
David J. P. Ferguson ◽  
Steven Abel ◽  
Alana Burrrell ◽  
Edward Rea ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Life Cycle ◽  
Live Cell Imaging ◽  
Malaria Parasite ◽  
Cell Imaging ◽  
Male Gamete ◽  
Live Cell ◽  
Basal Bodies ◽  
Parasite Transmission ◽  
Parasite Life Cycle

AbstractEukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not colocalise with kinetochores in the nucleus but instead revealed dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We thus uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

scholarly journals Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Joyce Jose ◽  
Aaron B. Taylor ◽  
Richard J. Kuhn
Keyword(s):  
Electron Microscopy ◽  
Life Cycle ◽  
Virus Replication ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Virus Production ◽  
Live Cell ◽  
Persistently Infected ◽  
Mosquito Cells

ABSTRACT Sindbis virus (SINV [genus Alphavirus , family Togaviridae ]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels. IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and assembly in the invertebrate vector and mechanisms producing two disease outcomes in two types of cells are yet to be identified. Using transmission electron microscopy and live-cell imaging with dual fluorescent protein-tagged SINV, we show that while insect and mammalian cells display similarities in entry and exit, they present distinct spatial and temporal organizations in virus replication and assembly. By characterizing acutely and persistently infected cells, we provide new insights into alphavirus replication and assembly in two distinct hosts, resulting in high-titer virus production in mammalian cells and continuous virus production at reduced levels in mosquito cells—presumably a prerequisite for alphavirus maintenance in nature.

scholarly journals Kinesin-8B controls basal body function and flagellum formation and is key to malaria transmission

2019 ◽  
Vol 2 (4) ◽  
pp. e201900488 ◽  
Author(s):  
Mohammad Zeeshan ◽  
David JP Ferguson ◽  
Steven Abel ◽  
Alana Burrrell ◽  
Edward Rea ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Life Cycle ◽  
Gene Targeting ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Male Gamete ◽  
Live Cell ◽  
Basal Bodies ◽  
Parasite Transmission ◽  
Parasite Life Cycle

Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division in male gametocyte together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated, but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy, and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B–GFP did not co-localise with kinetochores in the nucleus but instead revealed a dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We, thus, uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

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