Immuno Tomography (IT) and Imaging Mass Cytometry (IMC) for constructing spatially resolved, multiplexed 3D IMC data sets.

Purpose: We have previously used Immuno Tomography (IT) to identify label-retaining stem cell populations in the cornea and meibomian gland. While this method provides the unique ability to quantify stem cell populations comprised of 1-4 cells, the number of antigens that can be sequentially used to characterize these unique cells is limited by antigen stability after antibody stripping and re-probing. To address this deficiency, we have evaluated the capability of Imaging Mass Cytometry™ (IMC™) to generate multiplexed images using metal-conjugated antibodies to label IT plastic sections and generate 3-dimensional IMC data sets (3D IMC).

Aggresome assembly at the centrosome is driven by CP110-CEP97-CEP290 and centriolar satellites.

Protein degradation is critical to maintaining cellular homeostasis, and perturbation of the ubiquitin proteasome system leads to the accumulation of protein aggregates. These aggregates are either directed towards autophagy for destruction or sequestered into an inclusion, termed the aggresome, at the centrosome. Utilizing high-resolution quantitative analysis, here, we define aggresome assembly at the centrosome in human cells.

Spatial and proteomic profiling reveals centrosome-independent features of centriolar satellites.

Centriolar satellites are small electron-dense granules that cluster in the vicinity of centrosomes. Satellites have been implicated in multiple critical cellular functions including centriole duplication, centrosome maturation, and ciliogenesis, but their precise composition and assembly properties have remained poorly explored. Here, we perform in vivo proximity-dependent biotin identification (BioID) on 22 human satellite proteins, to identify 2,113 high-confidence interactions among 660 unique polypeptides.

A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface.

The centrosome is the primary microtubule organizing center of the cells and templates the formation of cilia, thereby operating at a nexus of critical cellular functions. Here, we use proximity-dependent biotinylation (BioID) to map the centrosome-cilium interface; with 58 bait proteins we generate a protein topology network comprising >7,000 interactions. Analysis of interaction profiles coupled with high resolution phenotypic profiling implicates a number of protein modules in centriole duplication, ciliogenesis, and centriolar satellite biogenesis and highlights extensive interplay between these processes.

Proteomic analyses of a bi-lobed structure in Trypanosoma brucei.

The position and function of the Golgi apparatus are tightly coupled with the microtubule-organizing centers (MTOCs) that play important roles in cell growth and polarity. In the unicellular parasite Trypanosoma brucei, the single Golgi apparatus is adjacent to a novel, bilobed structure located at the proximal base of the flagellum, near to the basal body that nucleates the flagellar axoneme. The duplication and segregation of the bilobed structure are tightly coupled to the duplication and segregation of Golgi, ER exit site, basal bodies, and flagellum, suggesting a role of this unique structure in the precise positioning, biogenesis, and inheritance of these single-copied structures during the cell cycle of procyclic T.

Biochemical characterization of the bi-lobe reveals a continuous structural network linking the bi-lobe to other single-copied organelles in Trypanosoma brucei.

Trypanosoma brucei, a unicellular parasite, contains several single-copied organelles that duplicate and segregate in a highly coordinated fashion during the cell cycle. In the procyclic stage, a bi-lobed structure is found adjacent to the single ER exit site and Golgi apparatus, forming both stable and dynamic association with other cytoskeletal components including the basal bodies that seed the flagellum and the flagellar pocket collar that is critical for flagellar pocket biogenesis. To further understand the bi-lobe and its association with adjacent organelles, we performed proteomic analyses on the immunoisolated bi-lobe complex.

An interplay between Centrin2 and Centrin4 on the bi-lobed structure in Trypanosoma brucei.

Centrins are conserved calcium-binding proteins important for various regulatory functions. In procyclic Trypanosoma brucei, TbCentrin2 and TbCentrin4 have distinct effects on cell division but both localize to the basal bodies that seed the flagellum, and a bi-lobed structure important for organelle duplication and cell division. Here we show that TbCentrin2 and TbCentrin4 both bind to the basal bodies and bi-lobed structure through the conserved C-terminal domain.

A comparative proteomic analysis reveals a new bi-lobe protein required for bi-lobe duplication and cell division in Trypanosoma brucei.

A Golgi-associated bi-lobed structure was previously found to be important for Golgi duplication and cell division in Trypanosoma brucei. To further understand its functions, comparative proteomics was performed on extracted flagellar complexes (including the flagellum and flagellum-associated structures such as the basal bodies and the bi-lobe) and purified flagella to identify new bi-lobe proteins. A leucine-rich repeats containing protein, TbLRRP1, was characterized as a new bi-lobe component.