scholarly journals The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

1999 ◽  
Vol 147 (2) ◽  
pp. 351-366 ◽  
Author(s):  
Vicki Mountain ◽  
Calvin Simerly ◽  
Louisa Howard ◽  
Asako Ando ◽  
Gerald Schatten ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cultured Cells ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Cross Linking ◽  
Simultaneous Inhibition ◽  
Balance Of Forces ◽  
And Function

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.

scholarly journals The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

2021 ◽  
Vol 22 (7) ◽  
pp. 3700
Author(s):  
Junna Hayashi ◽  
Jennifer Ton ◽  
Sparsh Negi ◽  
Daniel E. K. M. Stephens ◽  
Dean L. Pountney ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein Aggregation ◽  
Molecular Chaperone ◽  
Cross Linking ◽  
Αb Crystallin ◽  
The Cross ◽  
Shock Proteins ◽  
And Function

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.

scholarly journals Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium.

1996 ◽  
Vol 132 (3) ◽  
pp. 335-344 ◽  
Author(s):  
H Aizawa ◽  
K Sutoh ◽  
I Yahara
Keyword(s):  
Actin Filaments ◽  
Cell Movement ◽  
Structure And Function ◽  
Cross Linking ◽  
Low Molecular Weight ◽  
Filamentous Actin ◽  
Membrane Ruffling ◽  
And Function

Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d-cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co-localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.

scholarly journals Acentrosomal spindle assembly and stability in C. elegans oocytes requires a kinesin-12 non-motor microtubule interaction domain

2021 ◽  
Author(s):  
Ian Daniel Wolff ◽  
Jeremy Alden Hollis ◽  
Sarah Marie Wignall
Keyword(s):  
Adaptor Protein ◽  
Direct Interaction ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Interaction Domain ◽  
Microtubule Binding ◽  
C Elegans ◽  
Insight Into

During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in C. elegans oocytes, where kinesin-5 is not required to generate outward force. Instead, the kinesin-12 family motor KLP-18 performs this function. KLP-18 acts with adaptor protein MESP-1 (meiotic spindle 1) to sort microtubule minus ends to the periphery of a microtubule array, where they coalesce into spindle poles. If either of these proteins is depleted, outward sorting of microtubules is lost and minus ends converge to form a monoaster. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which these proteins collaborate to promote acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and MESP-1 activates non-motor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.

Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues

1999 ◽  
Vol 112 (8) ◽  
pp. 1149-1158 ◽  
Author(s):  
K. Hayashi ◽  
S. Yonemura ◽  
T. Matsui ◽  
S. Tsukita
Keyword(s):  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Tissue Type ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Erm Proteins

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

scholarly journals Pancreatic-Duodenal Homeobox 1 Regulates Expression of Liver Receptor Homolog 1 during Pancreas Development

2003 ◽  
Vol 23 (19) ◽  
pp. 6713-6724 ◽  
Author(s):  
Jean-Sébastien Annicotte ◽  
Elisabeth Fayard ◽  
Galvin H. Swift ◽  
Lars Selander ◽  
Helena Edlund ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Regulatory Region ◽  
Mobility Shift ◽  
Pancreatic Development ◽  
Downstream Target ◽  
Regulatory Cascade ◽  
And Function ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Liver receptor homolog 1 (LRH-1) and pancreatic-duodenal homeobox 1 (PDX-1) are coexpressed in the pancreas during mouse embryonic development. Analysis of the regulatory region of the human LRH-1 gene demonstrated the presence of three functional binding sites for PDX-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed that PDX-1 bound to the LRH-1 promoter, both in cultured cells in vitro and during pancreatic development in vivo. Retroviral expression of PDX-1 in pancreatic cells induced the transcription of LRH-1, whereas reduced PDX-1 levels by RNA interference attenuated its expression. Consistent with direct regulation of LRH-1 expression by PDX-1, PDX-1−/− mice expressed smaller amounts of LRH-1 mRNA in the embryonic pancreas. Taken together, our data indicate that PDX-1 controls LRH-1 expression and identify LRH-1 as a novel downstream target in the PDX-1 regulatory cascade governing pancreatic development, differentiation, and function.

scholarly journals Applications of novel bioreactor technology to enhance the viability and function of cultured cells and tissues

2020 ◽  
Vol 10 (2) ◽  
pp. 20190090 ◽  
Author(s):  
H. W. Hoyle ◽  
L. A. Smith ◽  
R. J. Williams ◽  
S. A. Przyborski
Keyword(s):  
Cultured Cells ◽  
Ex Vivo ◽  
Low Cost ◽  
Tissue Slices ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Tissue Constructs ◽  
And Function

As the field of tissue engineering continues to advance rapidly, so too does the complexity of cell culture techniques used to generate in vitro tissue constructs, with the overall aim of mimicking the in vivo microenvironment. This complexity typically comes at a cost with regards to the size of the equipment required and associated expenses. We have developed a small, low-cost bioreactor system which overcomes some of the issues of typical bioreactor systems while retaining a suitable scale for the formation of complex tissues. Herein, we have tested this system with three cell populations/tissues: the culture of hepatocellular carcinoma cells, where an improved structure and basic metabolic function is seen; the culture of human pluripotent stem cells, in which the cultures can form more heterogeneous tissues resembling the in vivo teratoma and ex vivo liver tissue slices, in which improved maintenance of cellular viability is seen over the 3 days tested. This system has the flexibility to be used for a variety of further uses and has the potential to provide a more accessible alternative to current bioreactor technologies.

scholarly journals Aurora A contributes to p150glued phosphorylation and function during mitosis

2010 ◽  
Vol 189 (4) ◽  
pp. 651-659 ◽  
Author(s):  
Pierre Romé ◽  
Emilie Montembault ◽  
Nathalie Franck ◽  
Aude Pascal ◽  
David M. Glover ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Microtubule Binding ◽  
Microtubule Binding Domain ◽  
Spindle Microtubules ◽  
And Function

Aurora A is a spindle pole–associated protein kinase required for mitotic spindle assembly and chromosome segregation. In this study, we show that Drosophila melanogaster aurora A phosphorylates the dynactin subunit p150glued on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A's function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the p150glued. Although wild-type p150glued binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous p150glued. Our results suggest that aurora A kinase participates in vivo to the phosphoregulation of the p150glued MBD to limit the microtubule binding of the dynein–dynactin complex and thus regulates spindle assembly.

Growth and characterization of porcine urinary bladder epithelial cells in vitro

1994 ◽  
Vol 266 (2) ◽  
pp. F298-F308 ◽  
Author(s):  
C. Guhe ◽  
W. Follmann
Keyword(s):  
Epithelial Cells ◽  
Urinary Bladder ◽  
Cultured Cells ◽  
Apical Cell ◽  
Membrane Integrity ◽  
Membrane Vesicles ◽  
Lactate Dehydrogenase Activity ◽  
And Function

Dividing long-term monolayer cultures of porcine urinary bladder epithelial cells were obtained by a combined mechanical and enzymatic isolation method. The serum-free cultured cells were investigated morphologically and characterized according to their growth characteristics and enzymatic functions. Investigations over a period up to 12 wk demonstrated that the cells regain their in vivo polarization with apically situated membrane vesicles and tight junctions between neighboring cells when they have built up a confluent monolayer. Activities of most marker enzymes for cell-differentiated status and function of such cells observed over a period of 4 wk in culture were conserved compared with the original tissue. Lactate dehydrogenase activity release into the medium was at low levels (< or = 5% of the total amount), indicating a good membrane integrity and cell viability. The chromosome set (2n = 38) did not change significantly during the first 5 wk, but, with additional culture time, the degree of polyploid and polynucleated cells increased comparable to the in vivo situation, in which the apical cell layer of the bladder mucosa also showed a high degree of polynucleation and polyploidy, indicative of a senescence process.

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