scholarly journals Live cell imaging of the assembly, disassembly, and actin cable–dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae

2004 ◽  
Vol 167 (3) ◽  
pp. 519-530 ◽  
Author(s):  
Thomas M. Huckaba ◽  
Anna Card Gay ◽  
Luiz Fernando Pantalena ◽  
Hyeong-Cheol Yang ◽  
Liza A. Pon
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Retrograde Flow ◽  
Yeast Saccharomyces Cerevisiae ◽  
Actin Cable ◽  
Complex Mutation ◽  
Conveyor Belts ◽  
Actin Patch ◽  
Mother Cells ◽  
Actin Cables

Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64–labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement. Rather, linear actin patch movement occurs at the same velocity and direction as the movement of actin cables. Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement. Our studies support a mechanism whereby actin cables serve as “conveyor belts” for retrograde movement and delivery of actin patches/endosomes to FM4-64–labeled internal compartments.

scholarly journals F-Actin Dynamics in Neurospora crassa

2010 ◽  
Vol 9 (4) ◽  
pp. 547-557 ◽  
Author(s):  
Adokiye Berepiki ◽  
Alexander Lichius ◽  
Jun-Ya Shoji ◽  
Jens Tilsner ◽  
Nick D. Read
Keyword(s):  
Neurospora Crassa ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Content Type ◽  
Actin Cables ◽  
Germ Tubes

ABSTRACT This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.

scholarly journals Microtubule retrograde flow retains neuronal polarization in a fluctuating state

2021 ◽  
Author(s):  
Max Schelski ◽  
Frank Bradke
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Retrograde Flow ◽  
Microtubule Array ◽  
Axon Extension ◽  
Pharmacological Manipulations ◽  
Neuronal Polarization

In developing vertebrate neurons, a neurite is formed by more than a hundred microtubules. While individual microtubules are dynamic, the microtubule array itself has been regarded as stationary. Using live cell imaging in combination with photoconversion techniques and pharmacological manipulations, we uncovered that the microtubule array flows retrogradely within neurites to the soma. This microtubule retrograde flow drives cycles of microtubule density, a hallmark of the fluctuating state before axon formation. Shortly after axon formation, microtubule retrograde flow slows down in the axon, which stabilizes microtubule density cycles and thereby functions as a molecular wedge to enable axon extension. We propose microtubule retrograde flow and its specific slowdown in the axon to be the long-sought mechanism to single one neurite out to drive neuronal polarization.

scholarly journals Live Cell Imaging of Mitochondrial Movement along Actin Cables in Budding Yeast

2004 ◽  
Vol 14 (22) ◽  
pp. 1996-2004 ◽  
Author(s):  
Kammy L. Fehrenbacher ◽  
Hyeong-Cheol Yang ◽  
Anna Card Gay ◽  
Thomas M. Huckaba ◽  
Liza A. Pon
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Live Cell ◽  
Mitochondrial Movement ◽  
Actin Cables

scholarly journals Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast

2005 ◽  
Vol 170 (4) ◽  
pp. 637-648 ◽  
Author(s):  
Vladimir Sirotkin ◽  
Christopher C. Beltzner ◽  
Jean-Baptiste Marchand ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Live Cell Imaging ◽  
Biochemical Analysis ◽  
Cell Imaging ◽  
Activator Proteins ◽  
Myosin I ◽  
Class 1 ◽  
Dynamic Structures ◽  
Actin Patch

Yeast actin patches are dynamic structures that form at the sites of cell growth and are thought to play a role in endocytosis. We used biochemical analysis and live cell imaging to investigate actin patch assembly in fission yeast Schizosaccharomyces pombe. Patch assembly proceeds via two parallel pathways: one dependent on WASp Wsp1p and verprolin Vrp1p converges with another dependent on class 1 myosin Myo1p to activate the actin-related protein 2/3 (Arp2/3) complex. Wsp1p activates Arp2/3 complex via a conventional mechanism, resulting in branched filaments. Myo1p is a weaker Arp2/3 complex activator that makes unstable branches and is enhanced by verprolin. During patch assembly in vivo, Wsp1p and Vrp1p arrive first independent of Myo1p. Arp2/3 complex associates with nascent activator patches over 6–9 s while remaining stationary. After reaching a maximum concentration, Arp2/3 complex patches move centripetally as activator proteins dissociate. Genetic dependencies of patch formation suggest that patch formation involves cross talk between Myo1p and Wsp1p/Vrp1p pathways.

scholarly journals Spatial telomere organization and clustering in yeast Saccharomyces cerevisiae nucleus is generated by a random dynamics of aggregation–dissociation

2013 ◽  
Vol 24 (11) ◽  
pp. 1791-1800 ◽  
Author(s):  
Nathanaël Hozé ◽  
Myriam Ruault ◽  
Carlo Amoruso ◽  
Angela Taddei ◽  
David Holcman
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Control Mechanism ◽  
Random Motion ◽  
Regulatory Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Diploid Cells ◽  
Different Levels ◽  
Telomere Dynamics

Spatial and temporal behavior of chromosomes and their regulatory proteins is a key control mechanism in genomic function. This is exemplified by the clustering of the 32 budding yeast telomeres that form foci in which silencing factors concentrate. To uncover the determinants of telomere distribution, we compare live-cell imaging with a stochastic model of telomere dynamics that we developed. We show that random encounters alone are inadequate to produce the clustering observed in vivo. In contrast, telomere dynamics observed in vivo in both haploid and diploid cells follows a process of dissociation–aggregation. We determine the time that two telomeres spend in the same cluster for the telomere distribution observed in cells expressing different levels of the silencing factor Sir3 protein, limiting for telomere clustering. We conclude that telomere clusters, their dynamics, and their nuclear distribution result from random motion, aggregation, and dissociation of telomeric regions, specifically determined by the amount of Sir3.

A Role for GEA1 and GEA2 in the Organization of the Actin Cytoskeleton in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 165 (3) ◽  
pp. 985-995
Author(s):  
Ewa Zakrzewska ◽  
Marjorie Perron ◽  
André Laroche ◽  
Dominick Pallotta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytoskeleton Organization ◽  
Actin Cable ◽  
Multicopy Suppressors ◽  
Actin Cytoskeleton Organization ◽  
Actin Cables ◽  
Adp Ribosylation Factor

Abstract Profilin is an actin monomer-binding protein implicated in the polymerization of actin filaments. In the budding yeast Saccharomyces cerevisiae, the pfy1-111 rho2Δ double mutant has severe growth and actin cytoskeletal defects. The GEA1 and GEA2 genes, which code for paralog guanosine exchange factors for Arf proteins, were identified as multicopy suppressors of the mutant phenotype. These two genes restored the polarized distribution of actin cortical patches and produced visible actin cables in both the pfy1-111 rho2Δ and pfy1Δ cells. Thus, overexpression of GEA1 or GEA2 bypassed the requirement for profilin in actin cable formation. In addition, gea1 gea2 double mutants showed defects in budding and in actin cytoskeleton organization, while overexpression of GEA1 or GEA2 led to the formation of supernumerary actin cable-like structures in a Bni1p/Bnr1p-dependent manner. The ADP-ribosylation factor Arf3p may be a target of Gea1p/Gea2p, since overexpression of ARF3 partially suppressed the profilin-deficient phenotype and a deletion of ARF3 exacerbated the phenotype of a pfy1-111 mutant. Gea1p, Gea2p, Arf1p, and Arf2p but not Arf3p are known to function in vesicular transport between the endoplasmic reticulum and the Golgi. In this work, we demonstrate a role for Gea1p, Gea2p, and Arf3p in the organization of the actin cytoskeleton.

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 Short Tetracysteine Tags to β-Tubulin Demonstrate the Significance of Small Labels for Live Cell Imaging

2004 ◽  
Vol 15 (12) ◽  
pp. 5616-5622 ◽  
Author(s):  
Martin Andresen ◽  
Rita Schmitz-Salue ◽  
Stefan Jakobs
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Open Reading Frames ◽  
Host Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent ◽  
Β Tubulin

Genetically encoded tags are of fundamental importance for live cell imaging. We show that small tetracysteine (TetCys) tags can be highly advantageous for the functionality of the host protein compared with large fluorescent protein tags. One to three concatenated small TetCys tags as well as the large green fluorescent protein (GFP) were fused by integrative epitope tagging to the C terminus of β-tubulin (Tub2) in the budding yeast Saccharomyces cerevisiae. The increasing tag size correlated with functional interference to the host protein. Tub2 tagged with either 1×TetCys (10 amino acids [aa]) or 2×TetCys (20 aa) was able to substitute Tub2 in haploid cells. In contrast, C-terminal tagging of Tub2 with 3×TetCys (29 aa) or with GFP (244 aa) resulted in nonviable haploid cells. Cells expressing Tub2-1×TetCys or Tub2-2×TetCys were stained with FlAsH, which selectively binds to the TetCys-tag. The stained cells displayed dynamic FlAsH-labeled microtubules and low cellular background fluorescence. The presented approach to tag open reading frames (ORFs) at their native loci with very small TetCys-tags and the subsequent visualization of the tagged proteins in vivo can be extended in principle to any ORF in S. cerevisiae.

scholarly journals Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

2006 ◽  
Vol 175 (6) ◽  
pp. 957-969 ◽  
Author(s):  
Thomas M. Huckaba ◽  
Thomas Lipkin ◽  
Liza A. Pon
Keyword(s):  
Actin Polymerization ◽  
Budding Yeast ◽  
Type Ii ◽  
Retrograde Flow ◽  
Cortical Actin ◽  
Actin Networks ◽  
Actin Cable ◽  
Intracellular Movement ◽  
Actin Cables ◽  
Type Ii Myosin

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.

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