Real time imaging reveals a peroxisomal reticulum in living cells

2000 ◽  
Vol 113 (20) ◽  
pp. 3663-3671 ◽  
Author(s):  
M. Schrader ◽  
S.J. King ◽  
T.A. Stroh ◽  
T.A. Schroer
Keyword(s):  
Real Time ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Real Time Imaging ◽  
Peroxisomal Targeting ◽  
Microtubule Motor ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.

scholarly journals Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 peroxisomal targeting signal but does not confer full biological activity on Pex5p

2000 ◽  
Vol 346 (1) ◽  
pp. 177-184 ◽  
Author(s):  
Rachel K. SZILARD ◽  
Richard A. RACHUBINSKI
Keyword(s):  
Yarrowia Lipolytica ◽  
Tetratricopeptide Repeat ◽  
Binding Assays ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Tetratricopeptide Repeat Domain ◽  
Tpr Domain ◽  
Peroxisomal Targeting Signal

Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. The Yarrowia lipolytica pex5-1 mutant fails to import a subset of peroxisomal matrix proteins, including those with a type 1 peroxisomal targeting signal (PTS1). Pex5p family members interact with a PTS1 through their characteristic tetratricopeptide repeat (TPR) domain. We used binding assays in vitro to investigate the nature of the association of Y. lipolytica Pex5p (YlPex5p) with the PTS1 signal. A purified recombinant YlPex5p fusion protein interacted specifically, directly and autonomously with a protein terminating in a PTS1. Wild-type YlPex5p translated in vitro recognized functional PTS1s specifically. This activity is abrogated by the substitution of an aspartic residue for a conserved glycine residue in the TPR domain (G455D) of YlPex5p encoded by the pex5-1 allele. Deletion analysis demonstrated that an intact TPR domain of YlPex5p is necessary but not sufficient for both interaction with a PTS1 and functional complementation of a strain lacking YlPex5p.

scholarly journals The preprophase band-associated kinesin-14 OsKCH2 is a processive minus-end-directed microtubule motor

2017 ◽  
Author(s):  
Kuo-Fu Tseng ◽  
Pan Wang ◽  
Yuh-Ru Julie Lee ◽  
Joel Bowen ◽  
Allison M. Gicking ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Preprophase Band ◽  
Cytoplasmic Dynein ◽  
Coiled Coils ◽  
Land Plants ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Microtubule Motor

AbstractIn animals and fungi, cytoplasmic dynein is a processive motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1 but not CC2 forms the coiled-coil for OsKCH2 dimerization. Instead, CC2 functions to enable OsKCH2 processivity by enhancing its binding to microtubules. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.

Transport of microinjected proteins into peroxisomes of mammalian cells: inability of Zellweger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal

1992 ◽  
Vol 12 (2) ◽  
pp. 531-541
Author(s):  
P A Walton ◽  
S J Gould ◽  
J R Feramisco ◽  
S Subramani
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Zellweger Syndrome ◽  
Fibroblast Cell ◽  
Artificial Hybrid ◽  
Peroxisomal Targeting ◽  
Hybrid Substrates ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

Previous work has shown that the firefly (Photinus pyralis) luciferase contains a C-terminal peroxisomal targeting signal consisting of the tripeptide Ser-Lys-Leu. This report describes the microinjection of two proteins, (i) luciferase and (ii) albumin conjugated to a peptide ending in the sequence Ser-Lys-Leu, into mammalian cells grown in tissue culture. Following microinjection, incubation of the cells at 37 degrees C resulted in peroxisomal transport of these exogenous proteins into catalase-containing vesicles. The translocation was both time and temperature dependent. The transport could be inhibited by coinjection of synthetic peptides bearing various peroxisomal targeting signal motifs. These proteins could be transported into peroxisomes in normal human fibroblast cell lines but not in cell lines derived from patients with Zellweger syndrome. These results demonstrate that microinjection of peroxisomal proteins yields an authentic in vivo system with which to study peroxisomal transport. Furthermore, these results reveal that the process of peroxisomal transport does not involve irreversible modification of the protein, that artificial hybrid substrates can be transported and used as tools to study peroxisomal transport, and that the defect in Zellweger syndrome is indeed the inability to transport proteins containing the Ser-Lys-Leu targeting signal into the peroxisomal lumen.

scholarly journals Real-time imaging of mast cell degranulation in vitro and in vivo

2016 ◽  
Vol 479 (3) ◽  
pp. 517-522 ◽  
Author(s):  
Kayo Horiguchi ◽  
Soichiro Yoshikawa ◽  
Asuka Saito ◽  
Salma Haddad ◽  
Takuya Ohta ◽  
...  
Keyword(s):  
Mast Cell ◽  
Real Time ◽  
Mast Cell Degranulation ◽  
Real Time Imaging

scholarly journals A motor neuron disease–associated mutation in p150Glued perturbs dynactin function and induces protein aggregation

2006 ◽  
Vol 172 (5) ◽  
pp. 733-745 ◽  
Author(s):  
Jennifer R. Levy ◽  
Charlotte J. Sumner ◽  
Juliane P. Caviston ◽  
Mariko K. Tokito ◽  
Srikanth Ranganathan ◽  
...  
Keyword(s):  
Cell Death ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Mitotic Spindle ◽  
Cytoplasmic Dynein ◽  
Aggregate Formation ◽  
Microtubule Motor ◽  
Delayed Recovery

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo

2019 ◽  
Author(s):  
Hacer Karatas ◽  
Tamara Maric ◽  
Pier Luca D’Alessandro ◽  
Aleksey Yevtodiyenko ◽  
Thomas Vorherr ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Imaging ◽  
Peptide Uptake

In vitro and in vivo real-time imaging with ultrasonic limited diffraction beams

1993 ◽  
Vol 12 (4) ◽  
pp. 819-829 ◽  
Author(s):  
Jian-yu Lu ◽  
T.K. Song ◽  
R.R. Kinnick ◽  
J.F. Greenleaf
Keyword(s):  
Real Time ◽  
Real Time Imaging

Analyses in transfected cells and in vitro of a putative peroxisomal targeting signal of rat liver serine:pyruvate aminotransferase

2002 ◽  
Vol 118 (4) ◽  
pp. 321-328 ◽  
Author(s):  
Takuji Mizuno ◽  
Kouichi Ito ◽  
Chiharu Uchida ◽  
Masatoshi Kitagawa ◽  
Arata Ichiyama ◽  
...  
Keyword(s):  
Rat Liver ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal
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