Nanotechnology‐based molecular photoacoustic and photothermal flow cytometry platform for in‐vivo detection and killing of circulating cancer stem cells

In‐vivo multicolor photoacoustic (PA) flow cytometry for ultrasensitive molecular detection of the CD44+ circulating tumor cells (CTCs) is demonstrated on a mouse model of human breast cancer. Targeting of CTCs with stem‐like phenotype, which are naturally shed from parent tumors, was performed with functionalized gold and magnetic nanoparticles. Results in vivo were verified in vitro with a multifunctional microscope, which integrates PA, photothermal (PT), fluorescent and transmission modules. Magnet‐induced clustering of magnetic nanoparticles in individual cells significantly amplified PT and PA signals. The novel noninvasive platform, which integrates multispectral PA detection and PT therapy with a potential for multiplex targeting of many cancer biomarkers using multicolor nanoparticles, may prospectively solve grand challenges in cancer research for diagnosis and purging of undetectable yet tumor‐initiating cells in circulation before they form metastasis. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ∼30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ∼138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.     

Evidence of a Reduced and Modified Mitochondrial Protein Import Apparatus in Microsporidian Mitosomes

Microsporidia are a group of highly adapted obligate intracellular parasites that are now recognized as close relatives of fungi. Their adaptation to parasitism has resulted in broad and severe reduction at (i) a genomic level by extensive gene loss, gene compaction, and gene shortening; (ii) a biochemical level with the loss of much basic metabolism; and (iii) a cellular level, resulting in lost or cryptic organelles. Consistent with this trend, the mitochondrion is severely reduced, lacking ATP synthesis and other typical functions and apparently containing only a fraction of the proteins of canonical mitochondria. We have investigated the mitochondrial protein import apparatus of this reduced organelle in the microsporidian Encephalitozoon cuniculi and find evidence of reduced and modified machinery. Notably, a putative outer membrane receptor, Tom70, is reduced in length but maintains a conserved structure chiefly consisting of tetratricopeptide repeats. When expressed in Saccharomyces cerevisiae, EcTom70 inserts with the correct topology into the outer membrane of mitochondria but is unable to complement the growth defects of Tom70-deficient yeast. We have scanned genomic data using hidden Markov models for other homologues of import machinery proteins and find evidence of severe reduction of this system.Microsporidia are a eukaryotic group highly adapted as obligate intracellular parasites (31, 50). They infect a diverse range of vertebrate and invertebrate animal hosts. In humans they are the cause of a number of diseases (e.g., gastroenteritis, encephalitis, and hepatitis), having their greatest impact on immunocompromised individuals, notably in children with human immunodeficiency virus (14, 31). Microsporidia are most closely related to fungi, although their high level of specialization as intracellular parasites obscured this relationship for a long time (18, 25, 30). Gene phylogenies now firmly connect these two groups, although it remains uncertain whether microsporidia are sisters to the fungi or represent a lineage derived from within fungal diversity (21, 28).A clear adaptive response to parasitism in microsporidia has been a reduction in cellular complexity. This was first recognized at an ultrastructural level with the apparent lack of peroxisomes, flagella, stacked Golgi bodies, and mitochondria (31). This reductive evolution is mirrored at a genomic level, with microsporidia containing the smallest eukaryotic genomes described to date (28, 29). The complete genomic sequence from the human microsporidian parasite Encephalitozoon cuniculi reveals a genome of only ∼2.9 Mb containing approximately 2,000 genes, in contrast to the 6,000 genes found in the genome of the model fungus Saccharomyces cerevisiae. The minimal genome of E. cuniculi has been achieved through three mechanisms in concert: (i) gene loss, resulting in broad loss of biochemical pathways and capabilities, including much basic energy metabolism and numerous anabolic pathways; (ii) gene compaction with an average intergenic space of ∼130 bp; and (iii) gene shortening, with E. cuniculi genes being on average 14% shorter than their homologues in fungi such as S. cerevisiae (28, 45). Thus, microsporidian evolution has apparently been shaped by a very strong trend to eliminate superfluous molecular and biochemical complexity.Despite earlier suppositions that microsporidia lacked mitochondria, genome and expressed sequence tag data from microsporidia suggested the presence of several proteins typically targeted to this organelle (3, 19, 20, 24, 28, 38). Immunolocalization of a mitochondrial Hsp70 to small double membrane-bound organelles in Trachipleistophora hominis provided strong evidence for the existence of a mitochondrion in microsporidia, albeit a simplified organelle that lacks cisternae (48). Annotation of genomic data from E. cuniculi provided compelling matches for only 22 proteins implicated in mitochondrial function, suggesting that the metabolism of this relict mitochondrion (or mitosome) is also significantly reduced compared to that of canonical mitochondria (28). Further, no mitochondrial genome has been retained; thus, biogenesis of this organelle is wholly dependent on nucleus-encoded proteins. Based on these 22 proteins, a major role for the mitosome is iron-sulfur cluster assembly (22, 28). No genes have been found for ATP synthesis via oxidative phosphorylation, suggesting loss of this activity in mitosomes (28, 46). While it is likely that further mitosome-targeted proteins will be identified, it is clear that compared to mitochondria from fungal relatives, which are known to import ∼1,000 proteins (40, 44), microsporidian mitosomes represent organelles with highly reduced proteomes, a feature consistent with other traits of cellular reduction.The highly reduced state of the microsporidian mitosome, requiring only a fraction of the protein diversity of other mitochondria, presents an interesting case for studying organelle biogenesis—particularly the machinery for protein import of nucleus-encoded proteins. Mitochondrial protein import has been best characterized in fungi, and in these systems most proteins are imported via four major import complexes: a TOM (translocase of the outer mitochondrial membrane), a SAM (sorting and assembly machinery), and one of two TIMs (translocase of the inner mitochondrial membrane), TIM23 or TIM22 (see Fig. ​Fig.5A)5A) (5, 36). These complexes are broadly conserved throughout fungi as well as animals (15). Mitochondrial proteins can take one of several routes to the mitochondrion via this apparatus (5, 36). Broadly, soluble matrix proteins are recognized at the TOM complex by the receptor protein Tom20 through the binding of N-terminal presequences with characteristic features (1, 5, 7, 8, 36). These proteins are passed through the pore protein Tom40 of the TOM to the TIM23 complex and then driven into the mitochondrial matrix by way of the presequence translocase-associated motor (PAM) complex, where their presequences are subsequently removed. Some membrane proteins can also be released into the inner membrane from the TIM23 complex. Mitochondrial proteins that apparently lack such an extension, notably including many of the membrane proteins, are recognized by internal sequence elements. Tom70 has a greater role in recognizing these internal signals and thus the import of hydrophobic proteins (4, 11, 32, 39, 47). Such hydrophobic proteins are often bound by cytosolic molecular chaperones (Hsp70 and/or Hsp90) en route to the mitochondrion, and Tom70 is known to independently bind to both the chaperone and the substrate protein (7, 23, 33, 52). While a measure of substrate overlap between Tom20 and Tom70 occurs, the division of responsibility between these two receptors has likely evolved in response to the wide range of substrate proteins that must be imported into mitochondria and the need to handle this complexity.Open in a separate windowFIG. 5.Schematics of the protein import machinery and pathways in yeast mitochondria (A) and E. cuniculi mitosome (B) based on identified homologues of the general fungal/animal pathways. Protein components of the yeast system were all represented by HMMs used to search the microsporidian genomic data and represent the major presequence-dependent and presequence-independent pathways. Homologues identified in E. cuniculi indicate a severely reduced import apparatus utilizing elements of the presequence-independent pathway.For microsporidia little is known of the protein import apparatus for their relict mitochondrion, the mitosome. Has the very reduced organelle proteome, in concert with a genome-wide trend of the loss of redundant or superfluous genes, resulted in a smaller and/or derived import apparatus? In this study we have investigated the microsporidian mitosome protein import apparatus from E. cuniculi in order to evaluate how this apparatus has responded to the reduction in the number of proteins required to be imported and the overall radical reduction in the number and size of proteins encoded in the nuclear genome. A putative homologue of the outer membrane receptor protein Tom70 is of particular interest as the only receptor for the TOM complex and, given the known structure of Tom70 proteins, provides a highly informative example of how proteins can be shortened in the course of genome reduction.     

Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1^?/?Neil1^?/? mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1^?/? and Neil1^?/?. The pulmonary tumors contained, exclusively, activating GGT → GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT → GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1^?/? and Neil1^?/?Nth1^?/? mice. The high incidence of tumors in our Nth1^?/?Neil1^?/? mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.     

TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites

TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5′ ends of pan-edited RNAs than at their 3′ ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3′ to 5′ progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3′ ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA–RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3′ to 5′ progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.     

Nix is a selective autophagy receptor for mitochondrial clearance

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.