Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium
Genre
Journal ArticleDate
2017-12-01Author
Luo, WRuba, A
Takao, D
Zweifel, LP
Lim, RYH
Verhey, KJ
Yang, W
Subject
AnimalsAxoneme
Carrier Proteins
Cilia
Cytosol
Diffusion
Green Fluorescent Proteins
Kinesin
Mice
MicroscopyNIH 3T3 CellsProbabilityProtein TransportProteinsTubulin
Permanent link to this record
http://hdl.handle.net/20.500.12613/4851Metadata
Show full item recordDOI
10.1038/s41598-017-16103-zAbstract
© 2017 The Author(s). Transport of membrane and cytosolic proteins in primary cilia is thought to depend on intraflagellar transport (IFT) and diffusion. However, the relative contribution and spatial routes of each transport mechanism are largely unknown. Although challenging to decipher, the details of these routes are essential for our understanding of protein transport in primary cilia, a critically affected process in many genetic diseases. By using a high-speed virtual 3D super-resolution microscopy, we have mapped the 3D spatial locations of transport routes for various cytosolic proteins in the 250-nm-wide shaft of live primary cilia with a spatiotemporal resolution of 2 ms and <16 nm. Our data reveal two spatially distinguishable transport routes for cytosolic proteins: an IFT-dependent path along the axoneme, and a passive-diffusion route in the axonemal lumen that escaped previous studies. While all cytosolic proteins tested primarily utilize the IFT path in the anterograde direction, differences are observed in the retrograde direction where IFT20 only utilizes IFT, and approximately half of KIF17 and one third of α-tubulin utilizes diffusion besides IFT.Citation to related work
Springer Science and Business Media LLCHas part
Scientific ReportsADA compliance
For Americans with Disabilities Act (ADA) accommodation, including help with reading this content, please contact scholarshare@temple.eduScopus Count
Collections
Except where otherwise noted, this item's license is described as CC BY
Related items
Showing items related by title, author, creator and subject.
-
Quantifying Nucleoporin Stoichiometry Inside Single Nuclear Pore Complexes In vivo© 2015, Nature Publishing Group. All rights reserved. The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ∼30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10-15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells.
-
A Spiroligomer α-Helix Mimic That Binds HDM2, Penetrates Human Cells and Stabilizes HDM2 in Cell CultureWe demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers [1]. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop. © 2012 Brown et al.
-
The retinoblastoma family: Twins or distant cousins?The destiny of a cell - whether it undergoes division, differentiation or death - results from an intricate balance of many regulators, including oncoproteins, tumor-suppressor proteins and cell-cycle-associated proteins. One of the better-studied tumor suppressors is the retinoblastoma protein, known as pRb or p105. Two recently identified proteins, pRb2/p130 and p107, show structural and functional similarities to pRb, and these proteins and their orthologs make up the retinoblastoma (Rb) family. Members of the family have been found in animals and plants, and a related protein is known in the alga Chlamydomonas. Members of the Rb family are bound and inactivated by viral proteins and, in turn, bind cellular transcription factors and repress their function, and can also form complexes with cyclins and cyclin-dependent kinases and with histone deacetylases. The are found in the nucleus and their subnuclear localization depends on binding to the nuclear matrix. Members of the family form part of a signal-transduction pathway called the Rb pathway, which is important in cell-cycle regulation and have roles in growth suppression, differentiation and apoptosis in different organisms and cell types.
