Laser-guidance based cell detection for identifying malignant cancerous cells without any fluorescent markers

Laser guidance technique employs the optical forces generated from a focused Gaussian laser beam incident on a biological cell to trap and guide the cell along the laser propagation direction. The optical force, which determines the guidance speed, is dependent on the cellular characteristics of the cell being guided, such as size, shape, composition and morphology. Different cell populations or subpopulations can be detected without any fluorescent markers by measuring their guidance speeds. We found that cell guidance speeds were sensitive enough to monitor the subtle changes during the progression of mouse fibroblast cells from normal to cancerous phenotype. The results also demonstrated that this technique can effectively distinguish mouse mammary cancerous cells with different metastatic competence. Laser guidance technique can be used as a label-free cell detection method for basic cell biological investigation and cancer diagnosis.     

BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions

The finding that morphogens, signalling molecules that specify cell identity, also act as axon guidance molecules has raised the possibility that the mechanisms that establish neural cell fate are also used to assemble neuronal circuits. It remains unresolved, however, how cells differentially transduce the cell fate specification and guidance activities of morphogens. To address this question, we have examined the mechanism by which the Bone morphogenetic proteins (BMPs) guide commissural axons in the developing spinal cord. In contrast to studies that have suggested that morphogens direct axon guidance decisions using non-canonical signal transduction factors, our results indicate that canonical components of the BMP signalling pathway, the type I BMP receptors (BMPRs), are both necessary and sufficient to specify the fate of commissural neurons and guide their axonal projections. However, whereas the induction of cell fate is a shared property of both type I BMPRs, axon guidance is chiefly mediated by only one of the type I BMPRs, BMPRIB. Taken together, these results indicate that the diverse activities of BMP morphogens can be accounted for by the differential use of distinct components of the canonical BMPR complex.     

Dormancy of Solitary Metastatic Cells

After arriving in a secondary site metastatic cells either begin proliferating, undergo apoptosis or remain as solitary dormant cells. The process of metastasis, although dangerous, is extremely inefficient with the majority of the cells undergoing apoptosis and thus becoming clinically irrelevant. Of the cells that begin proliferating, the few that make it past the micrometastasis stage may be of immediate clinical relevance. Dormant cells, while not of immediate clinical concern, are believed to be at least in part responsible for cancer recurrence that can occur decades after apparently successful initial treatment. Dormant solitary cells are different from “dormant” micrometastases, in which active proliferation is balanced by apoptosis. The mechanisms of cell cycle regulation and the function of the molecules regulating this process are well understood. However, there is relatively little known about the mechanisms controlling cell cycle regulation and dormancy of solitary metastatic cells. There are several inherent difficulties impeding the study of solitary cells. This review paper will examine the models used in the study of dormant solitary metastatic cells, methods of imaging and studying these cells, the molecular mechanisms believed to be responsible for solitary cell dormancy, and finally the unique treatment challenges posed by these cells.     

Basement membrane remodeling guides cell migration and cell morphogenesis during development

Basement membranes (BMs) are thin, dense forms of extracellular matrix that underlie or surround most animal tissues. BMs are enormously complex and harbor numerous proteins that provide essential signaling, mechanical, and barrier support for tissues during their development and normal functioning. As BMs are found throughout animal tissues, cells frequently migrate, change shape, and extend processes along BMs. Although sometimes used only as passive surfaces by cells, studies in developmental contexts are finding that BMs are often actively modified to help guide cell motility and cell morphogenesis. Here, I provide an overview of recent work revealing how BMs are remodeled in remarkably diverse ways to direct cell migration, cell orientation, axon guidance, and dendrite branching events during animal development.     

Chronic fluid flow is an environmental modifier of renal epithelial function

Although solitary or sensory cilia are present in most cells of the body and their existence has been known since the sixties, very little is been known about their functions. One suspected function is fluid flow sensing- physical bending of cilia produces an influx of Ca(++), which can then result in a variety of activated signaling pathways. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a progressive disease, typically appearing in the 5(th) decade of life and is one of the most common monogenetic inherited human diseases, affecting approximately 600,000 people in the United States. Because ADPKD is a slowly progressing disease, I asked how fluid flow may act, via the primary cilium, to alter epithelial physiology during the course of cell turnover. I performed an experiment to determine under what conditions fluid flow can result in a change of function of renal epithelial tissue. A wildtype epithelial cell line derived the cortical collecting duct of a heterozygous offspring of the Immortomouse (Charles River Laboratory) was selected as our model system. Gentle orbital shaking was used to induce physiologically relevant fluid flow, and periodic measurements of the transepithelial Sodium current were performed. At the conclusion of the experiment, mechanosensitive proteins of interest were visualized by immunostaining. I found that fluid flow, in itself, modifies the transepithelial sodium current, cell proliferation, and the actin cytoskeleton. These results significantly impact the understanding of both the mechanosensation function of primary cilia as well as the understanding of ADPKD disease progression.     

The FGF8-related signals Pyramus and Thisbe promote pathfinding, substrate adhesion, and survival of migrating longitudinal gut muscle founder cells

Fibroblast growth factors (FGFs) frequently fulfill prominent roles in the regulation of cell migration in various contexts. In Drosophila, the FGF8-like ligands Pyramus (Pyr) and Thisbe (Ths), which signal through their receptor Heartless (Htl), are known to regulate early mesodermal cell migration after gastrulation as well as glial cell migration during eye development. Herein, we show that Pyr and Ths also exert key roles during the long-distance migration of a specific sub-population of mesodermal cells that migrate from the caudal visceral mesoderm within stereotypic bilateral paths along the trunk visceral mesoderm toward the anterior. These cells constitute the founder myoblasts of the longitudinal midgut muscles. In a forward genetic screen for regulators of this morphogenetic process we identified loss of function alleles for pyr. We show that pyr and ths are expressed along the paths of migration in the trunk visceral mesoderm and endoderm and act largely redundantly to help guide the founder myoblasts reliably onto and along their substrate of migration. Ectopically-provided Pyr and Ths signals can efficiently re-rout the migrating cells, both in the presence and absence of endogenous signals. Our data indicate that the guidance functions of these FGFs must act in concert with other important attractive or adhesive activities of the trunk visceral mesoderm. Apart from their guidance functions, the Pyr and Ths signals play an obligatory role for the survival of the migrating cells. Without these signals, essentially all of these cells enter cell death and detach from the migration substrate during early migration. We present experiments that allowed us to dissect the roles of these FGFs as guidance cues versus trophic activities during the migration of the longitudinal visceral muscle founders.     

Patterns of fibronectin gene expression and splicing during cell migration in chicken embryos

A variety of evidence suggests that fibronectin (FN) promotes cell migration during embryogenesis, and it has been suggested that the deposition of FN along migratory pathways may also play a role in cell guidance. In order to investigate such a role for FN, it is important to determine the relative contribution of migrating and pathway-forming cells to the FN in the migratory track, as any synthesis of FN by the migrating cells might be expected to mask guidance cues provided by the exogenous FN from pathway-forming cells. We have therefore used in situ hybridization to determine in developing chicken embryos the distribution and alternative splicing of FN mRNA during three different cell migrations known to occur through FN-rich environments; neural crest cell migration, mesenchymal cell migration in the area vasculosa and endocardial cushion cell migration in the heart. Our results show that trunk neural crest cells do not contain significant FN mRNA during their initial migration. In contrast, migrating mesenchymal cells of the area vasculosa and endocardial cushion cells both contain abundant FN mRNA. Furthermore, the FN mRNA in these migrating mesenchymal and endocardial cells appears to be spliced in a manner identical with that present in the cells adjacent to their pathways. This in vivo evidence for FN synthesis by migrating and pathway cells argues against a generalized role for exogenously produced FN as a guidance mechanism for cell migration.     

中华蜜蜂工蜂蕈形体胚后发育中神经胶质的形成模式

本研究通过形态解剖、免疫组织化学等技术,对中华蜜蜂Apis cerana cerana工蜂蕈形体胚后发育中神经胶质的形成过程进行了比较研究。结果表明：蕈形体中神经胶质增殖的高峰期集中在幼虫发育末期到蛹发育早期;在工蜂蕈形体的蕈体冠、蕈体柄以及小叶的发育过程中,神经胶质细胞往往先于神经纤维网出现在特定的区域,引导神经纤维网的形成。它们一方面规定了神经纤维网的边界和区域,为神经纤维网提供内部的分隔;另一方面也为神经纤维的移动提供特定的“路标"和靶向。与神经纤维网相关联的神经胶质的数量的持续增加,除了神经胶质的分裂增殖外,还有一部分来自于外部细胞体层的神经胶质的迁入。     

A novel cell force sensor for quantification of traction during cell spreading and contact guidance

In this work, we present a ridged, microfabricated, force sensor that can be used to investigate mechanical interactions between cells exhibiting contact guidance and the underlying cell culture substrate, and a proof-of-function evaluation of the force sensor performance. The substrates contain arrays of vertical pillars between solid ridges that were microfabricated in silicon wafers using photolithography and deep reactive ion etching. The spring constant of the pillars was measured by atomic force microscopy. For time-lapse experiments, cells were seeded on the pillared substrates and cultured in an on-stage incubator on a microscope equipped with reflected differential interference contrast optics. Endothelial cells (ECs) and fibroblasts were observed during attachment, spreading, and migration. Custom image analysis software was developed to resolve cell borders, cell alignment to the pillars and migration, displacements of individual pillars, and to quantify cell traction forces. Contact guidance classification was based on cell alignment and movement angles with respect to microfabricated ridges, as well as cell elongation. In initial investigations made with the ridged cell force sensor, we have observed contact guidance in ECs but not in fibroblast cells. A difference in maximal amplitude of mechanical forces was observed between a contact-guided and non-contact-guided, but mobile, EC. However, further experiments are required to determine the statistical significance of this observation. By chance, we observed another feature of cell behavior, namely a reversion of cell force direction. The direction of forces measured under rounded fibroblast cells changed from outwards during early cell attachment to inwards during further observation of the spreading phase. The range of forces measured under fibroblasts (up to 138 nN) was greater than that measured in EC (up to 57 nN), showing that the rigid silicon sensor is capable of resolving a large range of forces, and hence detection of differences in traction forces between cell types. These observations indicate proof-of-function of the ridged cell force sensor to induce contact guidance, and that the pillared cell force sensor constructed in rigid silicon has the necessary sensitivity to detect differences in traction force vectors between different cell phenotypes and morphologies.     

高摄食压力下球形棕囊藻凝聚体的形成

棕囊藻包含囊体和游离单细胞两种生活史阶段。囊体是棕囊藻藻华爆发时的优势形态,藻华衰退时囊体能够形成凝聚体,但是棕囊藻游离单细胞的凝聚体极少被发现。本次研究将球形棕囊藻单细胞和高密度的海洋弯曲甲藻共同培养,使球形棕囊藻的生长承受高摄食压力,通过观察摄食者和棕囊藻的生长、凝聚体的数量和形态,阐明单细胞凝聚体的形成以及与摄食压力的关系。当球形棕囊藻进入衰退期时,高摄食压力引发游离单细胞聚合形成凝聚体,无摄食压力情况下,单细胞不形成凝聚体。凝聚体由无鞭毛细胞组成,细胞排列紧密,近似球体。凝聚体形成伊始,凝聚体内部可见凝胶状物质将细胞互相粘结,并且粘附了纤维等物质。凝聚体的体积和粘附的细胞数量逐渐提高,细胞排列愈加紧密,凝聚体内部形态和结构不易分辨。凝聚体的形成有效保护了部分单细胞免受摄食压力的影响,减少了摄食死亡率。凝聚体的形成是球形棕囊藻面临高摄食压力时采取的主动的防御策略。球形棕囊藻能够频繁引发大规模藻华的原因可能在于其在生长的各个阶段中均具有优越的竞争策略。     

Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres

Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.     

Trafficking of Crumbs3 during Cytokinesis Is Crucial for Lumen Formation

Although lumen generation has been extensively studied through so-called cyst-formation assays in Madin-Darby canine kidney (MDCK) cells, an underlying mechanism that leads to the initial appearance of a solitary lumen remains elusive. Lumen formation is thought to take place at early stages in aggregates containing only a few cells. Evolutionarily conserved polarity protein complexes, namely the Crumbs, Par, and Scribble complexes, establish apicobasal polarity in epithelial cells, and interference with their function impairs the regulated formation of solitary epithelial lumina. Here, we demonstrate that MDCK cells form solitary lumina during their first cell division. Before mitosis, Crumbs3a becomes internalized and concentrated in Rab11-positive recycling endosomes. These compartments become partitioned in both daughter cells and are delivered to the site of cytokinesis, thus forming the first apical membrane, which will eventually form a lumen. Endosome trafficking in this context appears to depend on the mitotic spindle apparatus and midzone microtubules. Furthermore, we show that this early lumen formation is regulated by the apical polarity complexes because Crumbs3 assists in the recruitment of aPKC to the forming apical membrane and interference with their function can lead to the formation of a no-lumen or multiple-lumen phenotype at the two-cell stage.     

Modelling cell migration strategies in the extracellular matrix

The extracellular matrix (ECM) is a highly organised structure with the capacity to direct cell migration through their tendency to follow matrix fibres, a process known as contact guidance. Amoeboid cell populations migrate in the ECM by making frequent shape changes and have minimal impact on its structure. Mesenchymal cells actively remodel the matrix to generate the space in which they can move. In this paper, these different types of movement are studied through simulation of a continuous transport model. It is shown that the process of contact guidance in a structured ECM can spatially organise cell populations. Furthermore, when combined with ECM remodelling, it can give rise to cellular pattern formation in the form of "cell-chains" or networks without additional environmental cues such as chemoattractants. These results are applied to a simple model for tumour invasion where it is shown that the interactions between invading cells and the ECM structure surrounding the tumour can have a profound impact on the pattern and rate of cell infiltration, including the formation of characteristic "fingering" patterns. The results are further discussed in the context of a variety of relevant processes during embryonic and adult stages.     

球形棕囊藻游离单细胞的密度与囊体形成的关系研究

球形棕囊藻(Phaeocystis globosa Scherffel)主要以囊体形态形成赤潮,由单细胞向囊体形态的转变是赤潮爆发的关键。本研究推测囊体形成的前提是游离单细胞达到一定密度阈值,当密度低于该阈值时,囊体无法形成。基于此,本文探究了不同条件(温度、营养充气搅动、摄食压力、初始密度)下囊体形成时游离单细胞的密度。结果显示:不同培养条件下,囊体形成所需的游离单细胞密度不一致,但都达到了104cells/mL的数量级;稀释试验表明,利用f/2培养基稀释使游离单细胞的密度小于104cells/mL时,囊体不能形成,而密度大于104cells/mL的游离单细胞对照组,在24 h内便有囊体形成。总的来说,游离单细胞在高密度情况下更容易形成囊体。     

STOP and GO with NO: nitric oxide as a regulator of cell motility in simple brains

During the formation of the brain, neuronal cell migration and neurite extension are controlled by extracellular guidance cues. Here, I discuss experiments showing that the messenger nitric oxide (NO) is an additional regulator of cell motility. NO is a membrane permeant molecule, which activates soluble guanylyl cyclase (sGC) and leads to the formation of cyclic GMP (cGMP) in target cells. The analysis of specific cells types in invertebrate models such as molluscs, insects and the medicinal leech provides insight how NO and cyclic nucleotides affect the wiring of nervous systems by regulating cell and growth-cone motility. Inhibition of the NOS and sGC enzymes combined with rescue experiments show that NO signalling orchestrates neurite outgrowth and filopodial dynamics, cell migration of enteric neurons, glial migration and axonogenesis of pioneer fibers. Cultured insect embryos are accessible model systems in which cellular mechanisms of NO-induced cytoskeletal reorganizations can be analyzed in natural settings. Finally, I will outline some indications that NO may also regulate cell motility in the developing and regenerating vertebrate nervous system.     

Novel brain wiring functions for classical morphogens: a role as graded positional cues in axon guidance

During embryonic development, morphogens act as graded positional cues to dictate cell fate specification and tissue patterning. Recent findings indicate that morphogen gradients also serve to guide axonal pathfinding during development of the nervous system. These findings challenge our previous notions about morphogens and axon guidance molecules, and suggest that these proteins, rather than having sharply divergent functions, act more globally to provide graded positional information that can be interpreted by responding cells either to specify cell fate or to direct axonal pathfinding. This review presents the roles identified for members of three prominent morphogen families--the Hedgehog, Wnt and TGFbeta/BMP families--in axon guidance, and discusses potential implications for the molecular mechanisms underlying their guidance functions.     

Gender Specific Brood Cells in the Solitary Bee Colletes halophilus (Hymenoptera; Colletidae)

We studied the reproductive behaviour of the solitary bee Colletes halophilus based on the variation in cell size, larval food amount and larval sex in relation to the sexual size dimorphism in this bee. Brood cells with female larvae are larger and contain more larval food than cells with males. Occasionally males are reared in female-sized cells. We conclude that a female C. halophilus in principal anticipates the sex of her offspring at the moment brood cell construction is started. Additionally a female is able to ‘change her mind’ about the sex of her offspring during a single brood cell cycle. We present a model that can predict the sex of the larvae in an early stage of development.     

Transforming growth factor beta responsiveness is modulated by the extracellular collagen matrix during hepatic ito cell culture

Hepatic sinusoidal Ito cells have the capacity to produce interstitial collagen types I and III as well as other matrix proteins and may be involved in hepatic fibrogenesis. Transforming growth factor beta (TGF beta) responsiveness was evaluated during in vitro cell culture, since increasing evidence suggests that this ubiquitous polypeptide can stimulate the production of collagenous proteins in a variety of cell types. TGF beta induced marked inhibition of Ito cell proliferation for cells grown on either a type I or a type IV collagen matrix. In marked contrast, the collagen synthetic response was considerably different for cells grown on a type I versus a type IV collagen matrix. When cells were grown on a type I collagen matrix, TGF beta caused a significant increase in the accumulation of collagen type I and III. When Ito cells were grown on a type IV collagen matrix, there was no stimulation of collagen production. TGF beta responsiveness was also evaluated in the setting of altered vitamin A concentrations. Freshly isolated Ito cells are engorged with vitamin A, the usual physiologic storage site for hepatic vitamin A. During in vitro culture and during in vivo fibrogenesis, Ito cells lose their vitamin A stores coincident with a transformation to a collagen-producing myofibroblast-like cell. When cultured Ito cells were grown on a type I collagen matrix and re-exposed to an increased concentration of vitamin A, the production of interstitial collagen was reduced. However, when the vitamin A-enriched Ito cells were exposed to TGF beta, the production of interstitial collagen was increased, similar to cells that had not received vitamin A.     

Microtubules and growth cone function

It has been recognized for a long time that the neuronal cytoskeleton plays an important part in neurite growth and growth cone pathfinding, the mechanism by which growing axons find an appropriate route through the developing embryo to their target cells. In the growth cone, many intracellular signaling pathways that are activated by guidance cues converge on the growth cone cytoskeleton and regulate its dynamics. Most of the research effort in this area has focussed on the actin, microfilament cytoskeleton of the growth cone, principally because it underlies growth cone motility, the extension and retraction of filopodia and lamellipodia, and these structures are the first to encounter guidance cues during growth cone advance. However, more recently, it has become apparent that the microtubule cytoskeleton also has a role in growth cone pathfinding and is also regulated by guidance cues operating through intracellular signaling pathways via engagement with cell membrane receptors. Furthermore, recent work has revealed an interaction between these two components of the growth cone cytoskeleton that is probably essential for growth cone turning, a fundamental growth cone behavior during pathfinding. In this short review I discuss recent experiments that uncover the function of microtubules in growth cones, how their behavior is regulated, and how they interact with the actin filaments.     

Molecular-scale topographic cues induce the orientation and directional movement of fibroblasts on two-dimensional collagen surfaces

Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.