scholarly journals Multiple kinesin-14 family members drive microtubule minus end–directed transport in plant cells

2017 ◽  
Vol 216 (6) ◽  
pp. 1705-1714 ◽  
Author(s):  
Moé Yamada ◽  
Yohko Tanaka-Takiguchi ◽  
Masahito Hayashi ◽  
Momoko Nishina ◽  
Gohta Goshima
Keyword(s):  
Physcomitrella Patens ◽  
Gene Knockout ◽  
Molecular Motor ◽  
Direct Interaction ◽  
Cell Types ◽  
Specific Class ◽  
Directed Transport ◽  
Genes Encoding ◽  
Acidic Phospholipids

Minus end–directed cargo transport along microtubules (MTs) is exclusively driven by the molecular motor dynein in a wide variety of cell types. Interestingly, during evolution, plants have lost the genes encoding dynein; the MT motors that compensate for dynein function are unknown. Here, we show that two members of the kinesin-14 family drive minus end–directed transport in plants. Gene knockout analyses of the moss Physcomitrella patens revealed that the plant-specific class VI kinesin-14, KCBP, is required for minus end–directed transport of the nucleus and chloroplasts. Purified KCBP directly bound to acidic phospholipids and unidirectionally transported phospholipid liposomes along MTs in vitro. Thus, minus end–directed transport of membranous cargoes might be driven by their direct interaction with this motor protein. Newly nucleated cytoplasmic MTs represent another known cargo exhibiting minus end–directed motility, and we identified the conserved class I kinesin-14 (ATK) as the motor involved. These results suggest that kinesin-14 motors were duplicated and developed as alternative MT-based minus end–directed transporters in land plants.

scholarly journals Multiple kinesin-14 family members drive microtubule minus-end-directed transport in plant cells

2017 ◽  
Author(s):  
Moé Yamada ◽  
Yohko Tanaka-Takiguchi ◽  
Masahito Hayashi ◽  
Momoko Nishina ◽  
Gohta Goshima
Keyword(s):  
Physcomitrella Patens ◽  
Gene Knockout ◽  
Molecular Motor ◽  
Direct Interaction ◽  
Cell Types ◽  
Specific Class ◽  
Directed Transport ◽  
Genes Encoding ◽  
Acidic Phospholipids

Minus-end-directed cargo transport along microtubules (MTs) is exclusively driven by the molecular motor dynein in a wide variety of cell types. Interestingly, plants have lost the genes encoding dynein during evolution; the MT motors that compensate for dynein function are unknown. Here, we show that two members of the kinesin-14 family drive minus-end-directed transport in plants. Gene knockout analyses of the moss Physcomitrella patens revealed that the plant-specific class-VI kinesin-14, KCBP, is required for minus-end-directed transport of the nucleus and chloroplasts. Purified KCBP directly bound to acidic phospholipids (PLs) and unidirectionally transported PL liposomes along MTs in vitro. Thus, minus-end-directed transport of membranous cargoes might be driven by their direct interaction with this motor protein. Newly nucleated cytoplasmic MTs represent another known cargo exhibiting minus-end-directed motility, and we identified the conserved class-I kinesin-14 (ATK) as the motor involved. These results suggest that kinesin-14 motors were duplicated and developed as alternative MT-based minus-end-directed transporters in land plants.

scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

scholarly journals HSP27 Is a Ubiquitin-Binding Protein Involved in I-κBα Proteasomal Degradation

2003 ◽  
Vol 23 (16) ◽  
pp. 5790-5802 ◽  
Author(s):  
Arnaud Parcellier ◽  
Elise Schmitt ◽  
Sandeep Gurbuxani ◽  
Daphné Seigneurin-Berny ◽  
Alena Pance ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Cell Types ◽  
26S Proteasome ◽  
Necrosis Factor Alpha ◽  
Polyubiquitin Chains ◽  
Ubiquitinated Proteins ◽  
Activation Of Transcription ◽  
Ubiquitin Proteasome

ABSTRACT HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-α). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-κB by degrading its main inhibitor, I-κBα. HSP27 overexpression increases NF-κB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, ΤNF-α, and interleukin 1β. HSP27 does not affect I-κBα phosphorylation but enhances the degradation of phosphorylated I-κBα by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-κBα. A protein complex that includes HSP27, phosphorylated I-κBα, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-κBα. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-κB activity.

scholarly journals The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p

2013 ◽  
Vol 202 (3) ◽  
pp. 509-526 ◽  
Author(s):  
David Shen ◽  
Hua Yuan ◽  
Alex Hutagalung ◽  
Avani Verma ◽  
Daniel Kümmel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Direct Interaction ◽  
Secretory Vesicles ◽  
Direct Role ◽  
Vesicular Traffic ◽  
Genes Encoding ◽  
Snare Motif ◽  
Liposome Fusion

A screen for mutations that affect the recruitment of the exocyst to secretory vesicles identified genes encoding clathrin and proteins that associate or colocalize with clathrin at sites of endocytosis. However, no significant colocalization of the exocyst with clathrin was seen, arguing against a direct role in exocyst recruitment. Rather, these components are needed to recycle the exocytic vesicle SNAREs Snc1p and Snc2p from the plasma membrane into new secretory vesicles where they act to recruit the exocyst. We observe a direct interaction between the exocyst subunit Sec6p and the latter half of the SNARE motif of Snc2p. An snc2 mutation that specifically disrupts this interaction led to exocyst mislocalization and a block in exocytosis in vivo without affecting liposome fusion in vitro. Overexpression of Sec4p partially suppressed the exocyst localization defects of mutations in clathrin and clathrin-associated components. We propose that the exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins. This could help to confer both specificity and directionality to vesicular traffic.

scholarly journals Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.

1994 ◽  
Vol 127 (4) ◽  
pp. 1049-1060 ◽  
Author(s):  
P D Kouklis ◽  
E Hutton ◽  
E Fuchs
Keyword(s):  
Intermediate Filaments ◽  
Transmembrane Domain ◽  
Direct Interaction ◽  
Cell Types ◽  
Epidermal Keratinocytes ◽  
Type I ◽  
Type Ii ◽  
Amino Terminal ◽  
Keratin Intermediate Filaments

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.

scholarly journals Malaria Pigment Hemozoin Impairs GM-CSF Receptor Expression and Function by 4-Hydroxynonenal

Antioxidants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1259
Author(s):  
Oleksii Skorokhod ◽  
Valentina Barrera ◽  
Giorgia Mandili ◽  
Federica Costanza ◽  
Elena Valente ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Cell Types ◽  
Receptor Expression ◽  
Amino Acid Residues ◽  
Gm Csf ◽  
Malaria Pigment ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
And Function

Malarial pigment hemozoin (HZ) generates the lipoperoxidation product 4-hydroxynonenal (4-HNE), which is known to cause dysregulation of the immune response in malaria. The inhibition of granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent differentiation of dendritic cells (DC) by HZ and 4-HNE was previously described in vitro, and the GM-CSF receptor (GM-CSF R) was hypothesised to be a primary target of 4-HNE in monocytes. In this study, we show the functional impact of HZ on GM-CSF R in monocytes and monocyte-derived DC by (i) impairing GM-CSF binding by 50 ± 9% and 65 ± 14%, respectively (n = 3 for both cell types); (ii) decreasing the expression of GM-CSF R functional subunit (CD116) on monocyte’s surface by 36 ± 11% (n = 6) and in cell lysate by 58 ± 16% (n = 3); and (iii) binding of 4-HNE to distinct amino acid residues on CD116. The data suggest that defective DC differentiation in malaria is caused by GM-CSF R dysregulation and GM-CSF R modification by lipoperoxidation product 4-HNE via direct interaction with its CD116 subunit.

scholarly journals A chimeric KaiA-like regulator extends the nonstandard KaiB3-KaiC3 clock system in bacteria

2021 ◽  
Author(s):  
Christin Koebler ◽  
Nicolas M. Schmelling ◽  
Alice Pawlowski ◽  
Philipp Spaet ◽  
Nina M. Scheurer ◽  
...  
Keyword(s):  
Environmental Changes ◽  
Response Regulator ◽  
Bacterial Species ◽  
Direct Interaction ◽  
Growth Conditions ◽  
Metabolic Switch ◽  
Functional Link ◽  
Genes Encoding ◽  
Rotation Of The Earth

The rotation of the Earth results in predictable environmental changes that pose challenges for organisms and force them to adapt. To address this daily rhythm, organisms from all kingdoms of life have evolved diverse timing mechanisms. In the cyanobacterium Synechococcus elongatus PCC 7942, the three proteins KaiA, KaiB, and KaiC constitute the central timing mechanism that drives circadian oscillations. In addition to the standard oscillator, named KaiAB1C1, Synechocystis sp. PCC 6803 harbors several, diverged clock homologs. The nonstandard KaiB3-KaiC3 system was suggested to impact the metabolic switch in response to darkness. Here, we demonstrate the direct interaction of KaiC3 with Sll0485, which is a potential new chimeric KaiA homolog that we named KaiA3. The existence of a functional link between these proteins is further supported by the co occurrence of genes encoding KaiA3 with the KaiB3-KaiC3-like gene products in 10 cyanobacterial and five other bacterial species. KaiA3 is annotated as a NarL-type response regulator due to its similarity to the response regulator receiver domains. However, its similarity to canonical NarL drastically decreases in the C-terminal domain, which resembles the circadian clock protein KaiA. In line with this, we detected the stimulation of KaiC3 phosphorylation by KaiA3 in vitro. Furthermore, we showed that deletion of the kaiA3 gene led to growth defects during mixotrophic growth conditions and, like a kaiC3-deficient mutant, viability was impaired during chemoheterotrophic growth in complete darkness. In summary, we suggest KaiA3 as a novel, nonstandard KaiA homolog within the cyanobacterial phylum, extending the KaiB3-KaiC3 system in Cyanobacteria and other prokaryotes.

Expression of the IGFBP-2 gene in post-implantation rat embryos

Development ◽  
1992 ◽  
Vol 114 (1) ◽  
pp. 59-66 ◽  
Author(s):  
T.L. Wood ◽  
R.D. Streck ◽  
J.E. Pintar
Keyword(s):  
Cell Types ◽  
Tail Bud ◽  
Igf Binding Proteins ◽  
Surface Ectoderm ◽  
Genes Encoding ◽  
Mantle Layer ◽  
Igf Binding ◽  
Post Implantation

The insulin-like growth factors (IGFs) stimulate mitogenesis in a variety of cell types both in vitro and in vivo. These effects are mediated by both IGF receptors and a family of IGF binding proteins (IGFBPs), which are found complexed with the IGFs in serum and tissue fluids. Here we compare the sites of expression during early rat embryogenesis of the genes encoding the RGD-containing IGF binding protein IGFBP-2 and IGF-II. At all ages from early post-implantation through mid-gestation, the expression of IGFBP-2 was highly complementary to IGF-II. IGFBP-2 mRNA was detected throughout the epiblast of the egg cylinder as early as e7, when IGF-II expression was restricted to trophectoderm and other extraembryonic cells. As gastrulation proceeded, IGFBP-2 expression ceased as IGF-II expression began in the newly formed embryonic and extra-embryonic mesoderm, but was retained in other epiblast derivatives including the surface ectoderm and neuroectoderm, throughout its rostral-caudal extent. By e10-e11, IGFBP-2 expression in neuroectoderm was restricted to the rostral brain of the primary neural tube and was found in the new population of neuroepithelium formed in the tail bud during secondary neurulation. IGFBP-2 expression remained high in the ventricular layer of the rostral brain into mid-gestation ages but decreased or disappeared as cells entered the mantle layer and began to express the neurofilament-related gene alpha-internexin. IGFBP-2 mRNA was abundant in surface ectoderm, particularly that of the branchial arches, and all ectodermal placodes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Myosin Va interacts with the exosomal protein spermine synthase

2019 ◽  
Vol 39 (3) ◽  
Author(s):  
Luciano G. Dolce ◽  
Rui M. P. Silva-Junior ◽  
Leandro H. P. Assis ◽  
Andrey F. Z. Nascimento ◽  
Jackeline S. Araujo ◽  
...  
Keyword(s):  
Molecular Motor ◽  
Direct Interaction ◽  
Tail Domain ◽  
Spermine Synthase ◽  
Cytoplasmic Vesicles ◽  
Myosin Va ◽  
Cold Shock Domain ◽  
Protein Components ◽  
Hybrid Screening

AbstractMyosin Va (MyoVa) is an actin-based molecular motor that plays key roles in the final stages of secretory pathways, including neurotransmitter release. Several studies have addressed how MyoVa coordinates the trafficking of secretory vesicles, but why this molecular motor is found in exosomes is still unclear. In this work, using a yeast two-hybrid screening system, we identified the direct interaction between the globular tail domain (GTD) of MyoVa and four protein components of exosomes: the WD repeat-containing protein 48 (WDR48), the cold shock domain-containing protein E1 (CSDE1), the tandem C2 domain-containing protein 1 (TC2N), and the enzyme spermine synthase (SMS). The interaction between the GTD of MyoVa and SMS was further validated in vitro and displayed a Kd in the low micromolar range (3.5 ± 0.5 µM). SMS localized together with MyoVa in cytoplasmic vesicles of breast cancer MCF-7 and neuroblastoma SH-SY5Y cell lines, known to produce exosomes. Moreover, MYO5A knockdown decreased the expression of SMS gene and rendered the distribution of SMS protein diffuse, supporting a role for MyoVa in SMS expression and targeting.

scholarly journals Coordinate defects in human histocompatibility leukocyte antigen class II expression and antigen presentation in bare lymphocyte syndrome.

1994 ◽  
Vol 179 (6) ◽  
pp. 2017-2022 ◽  
Author(s):  
S Kovats ◽  
S Drover ◽  
W H Marshall ◽  
D Freed ◽  
P E Whiteley ◽  
...  
Keyword(s):  
Antigen Presentation ◽  
Transcriptional Control ◽  
Class Ii ◽  
Functional Class ◽  
Specific Class ◽  
Protein Antigens ◽  
Bare Lymphocyte Syndrome ◽  
Peptide Antigens ◽  
Genes Encoding

The human immunodeficiency, type II bare lymphocyte syndrome (BLS), has been attributed to a defect in the transcription of class II histocompatibility genes. Immunocompetence, as assessed by functional exogenous antigen presentation, was not restored in immortalized B cells, derived from a BLS patient, after transfection with HLA-DR class II structural genes. Incubation of protein antigens, as well as infectious virus, with DR-transfected BLS cells failed to induce activation of antigen-specific helper T lymphocytes. Peptide antigens were presented by class II molecules displayed on BLS cells, although the conformation of these class II proteins was altered as indicated by epitope mapping. This defect in antigen presentation was independent of the specific class II DR allele transfected into BLS cells. Genetic complementation analysis has been used with BLS cells to demonstrate that the defect in class II gene transcription is linked to the absence of a trans-acting factor. Similarly, functional class II dimers were restored after in vitro fusion of cells derived from two distinct BLS complementation groups, implying that specific transcriptional control elements are shared by a gene critical for antigen presentation and genes encoding HLA class II antigens. Thus, two important functionally linked pathways of class II molecules, structural gene expression and antigen presentation, share a common regulatory pathway defective in BLS.

Sign in / Sign up

Export Citation Format

Share Document