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Find video protocols related to scientific articles indexed in Pubmed.
Mpl traffics to the cell surface through conventional and unconventional routes.
Traffic
PUBLISHED: 06-10-2014
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Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA-mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER-tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER-Golgi and autolysosome secretory pathways, as well as recycling.
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The architectural relationship of components controlling mast cell endocytosis.
J. Cell. Sci.
PUBLISHED: 08-28-2013
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Eukaryotic cells use multiple routes for receptor internalization. Here, we examine the topographical relationships of clathrin-dependent and clathrin-independent endocytic structures on the plasma membranes of leukemia-derived mast cells. The high affinity IgE receptor (Fc?RI) utilizes both pathways, whereas transferrin receptor serves as a marker for the classical clathrin-mediated endocytosis pathway. Both receptors were tracked by live-cell imaging in the presence or absence of inhibitors that established their differential dependence on specific endocytic adaptor proteins. The topology of antigen-bound Fc?RI, clathrin, dynamin, Arf6 and Eps15-positive structures were analyzed by 2D and 3D immunoelectron microscopy techniques, revealing their remarkable spatial relationships and unique geometry. We conclude that the mast cell plasma membrane has multiple specialized domains for endocytosis. Their close proximity might reflect shared components, such as lipids and adaptor proteins, that facilitate inward membrane curvature. Intersections between these specialized domains might represent sorting stations that direct cargo to specific endocytic pathways.
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Nested high-resolution melting curve analysis a highly sensitive, reliable, and simple method for detection of JAK2 exon 12 mutations--clinical relevance in the monitoring of polycythemia.
J Mol Diagn
PUBLISHED: 04-19-2011
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JAK2 exon 12 mutations are found in myeloproliferative disorders characterized by erythrocytosis. Lying in a 33-bp region and conserving the open reading frame, they often present a low allelic burden (<10%), which excludes screening with techniques such as allele-specific PCR or different sequencing protocols. High-resolution melting (HRM), a fast in-tube method, seems the most accurate routine technique for that. We describe a reliable and powerful nested HRM technique, independent of DNA preparation and with technical sensitivity of 100% (95% CI, 93% to 100%) and specificity of 96.7% (95% CI, 89.7% to 96.7%). Screening a cohort of 10 idiopathic erythrocytosis, 28 polycythemia vera, and 7 secondary erythrocytosis cases allowed the detection of 15 mutants, including 9 different mutations, of which 3 were unreported, all in the polycythemia vera group, and presented a characteristic profile: pure erythrocytosis associated with low serum erythropoietin. Threshold detection level ranged from 1% to 3% allelic burden, depending on the mutation. All of the HRM positive signals were found mutated by sequencing. Six of them (40%), however, required cloning before sequencing, because of low allelic burden. Classic techniques such as genomic sequencing may therefore miss patients with mutations. Given its sensitivity, HRM (and nested HRM) can be used in routine diagnosis and seems to be the most efficient of current techniques for detection of JAK2 exon 12 mutations.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.