The paper is quite interesting - I believe it is the first time to make filopodia like structures in vitro using Xenopus egg extracts. Really hope they did it with just purified proteins! I don't understand why they started with toca-1 in the mixture. The F-Bar induced membrane curvature may be the reason why the drawing in their model is like hair follicle. And, it is recruited first, even before actin! Their immunodepletion of toca-1 had minimal effect, and their explanation is that there are a lot of F-Bar proteins in the extract could substitute. The questions are:

• Is it possible that the F-Bar protein making those follicle like base first, which then recruits N-WASP? The recruitment of N-WASP might very well be the consequence of local lipid composition.
• Does the follicle like membrane structure restrict the branching direction and force the merging of the actin branches?
• Unlike the bead assay, minimal branched actin structure is observed with this FLS assay. But, the authors have not shown an EM image of these FLS bases (follicle region), where I believe the branches are...

FLS assays

To make the supported bilayers, No. 1.5 glass coverslips were incubated with freshly prepared liposomes containing 45 % PC, 45 % PI, and 10 % PI(4,5)P2 in XB buffer for 20 min at room temperature, followed by extensive washing with XB buffer. Membrane phase separation was variable and was largely influenced by the particular batch of glass. Rigorous washing of the coverslips with hot detergent also promoted the liquid disordered phase. All assays were carried out at room temperature (~22 °C).

For the purified system experiments, prenylated Cdc42.GTPrS was supplied to the lipid bilayer from 100 nM Cdc42-RhoGDI in solution using the EDTA exchange reaction (S9). The reaction mixture including N-WASP-WIP, toca-1, Arp2/3 complex, and actin as previously described (S2) was added after Cdc42 loading.

Typical FLS reactions (50 μl volume) contained a 2-fold dilution of Xenopus egg extract, 4 μM Alexa 647 actin (10% labeling efficiency, rabbit skeleton muscle actin), 0.35 M sucrose, 1 mM ATP, 1 mM MgCl2, 7.5 mM phosphocreatine in XB buffer. The reaction mixtures were added on top of the freshly prepared supported bilayer and monitored with a spinning disk confocal microscope.

For the pulse chase experiments, the second reaction (5 μl volume) Xenopus egg extract, 12 uM Alexa-488 actin (5% labeling efficiency, rabbit skeletal muscle actin), 1 mM ATP, 7.5 mM phosphocreatine in XB and 5 μl was added gently on top of the first reactions.

For dose response of FLS initial elongation, the reaction mixture was supplement with different dose of GST-CA and images were taken after 7 min.

For Arp2/3 complex independent elongation experiments, the first reaction was supplemented with 50 nM Alexa568-Arp2/3 complex. The second reaction is assembled similarly to the pulse-chase experiments with different doses of GST-CA. For the GFP-PH domain experiments, 50-300 nM was used.

For timelapse movies of FLS growth, an oxygen scavenger mix was added which contained: 4.5 mg/ml glucose, 0.5 % 2-mercaptoethanol, 0.2 mg/ml glucose oxidase (Sigma-Aldrich), 35 ug/ml catalase (Sigma-Aldrich).

Light microscopy

Microscopy for figures 1, 2 and 5 was performed using an inverted Nikon TE2000U microscope with a 100x, 1.4 NA Plan Apochromat objective lens and motorized stage and focus motor from Prior. Confocal images were obtained using a Yokogawa CSU-10 spinning disk confocal head with Prairie laser launch with a 2.5 W water-cooled Coherent Argon-Krypton laser. Excitation and emission wavelengths were selected and attenuated with an AOTF and a triple 488/568/647 dichroic mirror from Chroma. GFP and Alexa-488 were visualized using the 488 laser line and 525/50 emission filter; Alexa-568 was visualized using the 568 laser line and 600/45 emission filter; Alexa-647 was visualized by the 647 laser line and 700/75 emission filter (Chroma). Images were collected with a ORCA-AG cooled CCD camera from Hamamatsu and Metamorph software v7.6 (Molecular Devices). Exposure times were typically 100~400 ms using 25~50% laser power and a bin of 2x2. Z-stacks were collected with a step size of 0.5 μm.

Light microscopy for figure 3 and supplementary figures 2, 3 and 4 was performed using an inverted Nikon Ti-E microscope with a 100x, 1.4 NA Plan Apochromat objective lens and motorized stage from Prior. Confocal images were obtained using a Yokogawa CSU-10 spinning disk confocal head with 100 mW Argon-Krypton laser from Melles Griot. Excitation and emission wavelengths were selected using Sutter filter wheels and a triple 488/568/647 dichroic mirror from Chroma. Images were collected with an ORCA-ER cooled CCD camera from Hamamatsu and Metamorph software v7.6 (Molecular Devices). GFP was visualized using the 488 laser line selected with a 488/10 excitation filter and 525/50 emission filter; rhodamine and Alexa-568 were visualized using the 568 laser line selected with a 568/10 excitation filter and 620/60 emission filter; Alexa-647 was visualized by the 647 laser line selected with a 647/10 filter, and 647/10 emission filter (Chroma). Exposure times were typically 200 ms using a bin of 2x2.

For time-lapse experiments of FLS initiation, the Perfect Focus System (Nikon) was used to maintain focus, and images were acquired every 10 s for 10 minutes. Z-stacks were acquired with a step size of 1 μm. For the fluorescence recovery after photobleaching experiments of the supported bilayer, wide-field epifluorescence illumination was used (with a Hamamatsu ORCA-R2 cooled CCD camera and an X-Cite series 120 light source) and rhodamine-PE was photobleached to 80-90% of initial intensity with 515 nm light from a nitrogen pulse laser (Photonic Instruments Micropoint system) focused to a spot less than 1 micron in diameter. The filter was Y-2E/C (excitation: 560/40 dichroic: 595 emission; 630/60) from Nikon. The exposure time was 25 ms, and images were typically acquired every 1 s for 1-20 min.

For the multispectral total internal reflection fluorescence microscopy in figure 4, we used a Nikon Ti-E inverted motorized microscope with integrated Perfect Focus System, Nikon 100x 1.49 NA TIRF DIC objective lens, Nikon halogen trans illuminator with 0.52 NA LWD and 0.85 NA Dry condenser, Nikon dual-port TIRF/Epi illuminator with motorized laser incident angle adjustment and motorized switching between TIRF and epi-illumination. For lasers, a Solamere laser launch was used with 100mW 491nm, 75mW 561nm and 30mW 640nm solid state lasers with a fiber-optic delivery system and 4-channel AOTF. A Prior Proscan II controller was used for fast excitation and emission filter wheels, fast transmitted and epi-fluorescence light path shutters, and a linear-encoded motorized stage. A Chroma zet405/491/561/638 dichroic mirror was used with a 491 nm laser line and a 525/50 emission filter for GFP; a 561 laser line and 600/50 emission filter for Alexa568; and a 640 laser line and a 700/75 emission filter for Alexa647. In addition to emission filters, a custom Chroma laser notch filter was used in the emission path to further block the illumination light from reaching the camera and to minimize interference patterns. Images were collected with a Hamamatsu ImagEM 512x512 back-thinned electron multiplying cooled CCD camera and MetaMorph v7.7 (Molecular Devices). Exposure times were typically ~100 ms using 25~50% laser power.

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Rat iPS into mouse Pdx1-/-, very nice design!

Cell. 2010 Sep 3;142(5):787-99.
Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells.
Kobayashi T, Yamaguchi T, Hamanaka S, Kato-Itoh M, Yamazaki Y, Ibata M, Sato H, Lee YS, Usui J, Knisely AS, Hirabayashi M, Nakauchi H.

Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; Japan Science Technology Agency, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.

PMID: 20813264
DOI: 10.1016/j.cell.2010.07.039

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I am quite surprised to see this paper went Science.
It is a paper with nice cell biology images.

But, the molecular mechanism elucidated in the paper is far from satisfaction. Is it because all the developmental biologist doesn't care the molecular mechanism?

I speculate the reason it is a Science paper is because Figure 4E.

• Bardet–Biedl syndrome: wiki, ncbi
• Meckel syndrome: "While not precisely known, it is estimated that the general rate of incidence, according to Bergsma, for Meckel syndrome is 0.02 per 10,000 births. According to another study done six years later, the incidence rate could vary from 0.07 to 0.7 per 10,000 births. ... accounts for 5% of all neural tube defects in Finland."
• Why not perform some kind rescue experiments using those Fritz mutants?
• Fritz with Septins. I don't think the data to link them together are very strong in this paper / not sure about other evidences in the literature.

Any thought?

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Anyway, the two papers are...

Nature. 2010 Jul 19. [Epub ahead of print]

Epigenetic memory in induced pluripotent stem cells.

Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, Kim J, Aryee MJ, Ji H, Ehrlich LI, Yabuuchi A, Takeuchi A, Cunniff KC, Hongguang H, McKinney-Freeman S, Naveiras O, Yoon TJ, Irizarry RA, Jung N, Seita J, Hanna J, Murakami P, Jaenisch R, Weissleder R, Orkin SH, Weissman IL, Feinberg AP, Daley GQ.

Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Manton Center for Orphan Disease Research, Howard Hughes Medical Institute, Children's Hospital Boston and Dana Farber Cancer Institute; Division of Hematology, Brigham and Women's Hospital; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School; Harvard Stem Cell Institute; Boston, Massachusetts 02115, USA.

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

PMID: 20644535

Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells

Jose M Polo, Susanna Liu, Maria Eugenia Figueroa, Warakorn Kulalert, Sarah Eminli, Kah Yong Tan, Effie Apostolou, Matthias Stadtfeld, Yushan Li, Toshi Shioda, Sridaran Natesan, Amy J Wagers, Ari Melnick, Todd Evans & Konrad Hochedlinger

Nature Biotechnology (2010) DOI: 10.1038/nbt.1667

Received 26 March 2010 Accepted 09 July 2010 Published online 19 July 2010

Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.

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Nat Chem Biol. 2010 Feb;6(2):102-4. Epub 2009 Dec 20.

Chemical reprogramming of Caenorhabditis elegans germ cell fate.

Morgan CT, Lee MH, Kimble J.

Medical Scientist Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA.

Small molecules can control cell fate in vivo and may allow directed induction of desired cell types, providing an attractive alternative to transplant-based approaches in regenerative medicine. We have chemically induced functional oocytes in Caenorhabditis elegans adults that otherwise produced only sperm. These findings suggest that chemical approaches to therapeutic cell reprogramming may be feasible and provide a powerful platform for analyzing molecular mechanisms of in vivo cell reprogramming.

PMID: 20081824

Interesting points of the paper

• Cell fate reprogramming within organisms has the potential to revolutionize regenerative medicine
• Small molecules can modulate cell fates in vivo, and chemical induction of a desired cell type could provide an attractive alternative to transplant- or viral-based cell replacement therapies.
• Such continued cell fate specification in adults is typical of many vertebrate tissues, including blood and intestinal epithelium.
• After 24 h of treatment, virtually all mutants made oocytes (99%; n = 158).
• In summary, we have shown that germ cell fate can be chemically reprogrammed within adult C. elegans and that reprogramming can induce a cell type that was absent without treatment.
• We do not know whether the reprogramming occurs by lineage switching of progenitors or by direct conversion of spermatocytes to oocytes.
• Therefore, our findings provide a paradigm that may facilitate pharmacological approaches to therapeutic cellular reprogramming in other organisms.

Reviews on F1000

Robert K Herman
University of Minnesota, United States of America

The authors of this paper were able to rescue a Caenorhabditis elegans mutant and alter the fates of particular cells by treating the animals with a small molecule rather than by the more usual approach of gene therapy.

The germlines of wild-type C. elegans hermaphrodites first produce sperm and then switch to oocyte production. In this work, the authors make use of a double mutant that has increased mitogen-activated protein (MAP) kinase activity in its germline; as a consequence, the mutants are unable to switch to oocyte production and make only sperm. After exposure to any of three different small molecule inhibitors of MAP kinase activity, the mutants were able to reprogram some germ cells to make fully functional oocytes, which thereby rescued the sterility of the mutants. The authors showed that the oocytes were truly reprogrammed germ cells rather than the result of differential proliferation or blocked apoptosis of cells specified as oocytes in the mutant. MAP kinase plays multiple roles in worm development; the authors avoided serious side effects by limiting both the dose and time of exposure to the chemical. This work supports the idea that chemical reprogramming of cell fates may be useful in the treatment of some human diseases.

--

Andy Golden
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), United States of America

In this study, reprogramming germ cells to make a missing cell type has been accomplished in Caenorhabditis elegans by simply soaking animals in chemical inhibitors of the Ras/ERK/MEK pathway.

One goal of regenerative medicine is to provide cells that are missing or destroyed. One minimally invasive approach would be to reprogram endogenous cells into the desired cell type that is missing in a specific disease. Morgan et al. have successfully induced the production of functional oocytes in C. elegans hermaphrodite mutants that are sterile because they only make sperm. By treating adults with MAP kinase and MEK inhibitors, they were able to reprogram germ cell fates and generate oocytes, which were functional based on the observation that they were fertilized and developed into adults. This probably works well in C. elegans because the germline can be manipulated after all other tissues have been generated; MAP kinase inhibition at earlier times in development would have more drastic effects.

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Science. 2010 Mar 11. [Epub ahead of print]

Caspase-Dependent Conversion of Dicer Ribonuclease into a Death-Promoting Deoxyribonuclease.

Nakagawa A, Shi Y, Kage-Nakadai E, Mitani S, Xue D.

Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.

Chromosome fragmentation is a hallmark of apoptosis, conserved in diverse organisms. In mammals, caspases activate apoptotic chromosome fragmentation by cleaving and inactivating an apoptotic nuclease inhibitor. We report that inactivation of the Caenorhabditis elegans dcr-1 gene, which encodes the Dicer ribonuclease important for processing of small RNAs, compromises apoptosis and blocks apoptotic chromosome fragmentation. DCR-1 was cleaved by the CED-3 caspase to generate a C-terminal fragment with deoxyribonuclease activity, which produced 3' hydroxyl DNA breaks on chromosomes and promoted apoptosis. Thus, caspase-mediated activation of apoptotic DNA degradation is conserved. DCR-1 functions in fragmenting chromosomal DNA during apoptosis, in addition to processing of small RNAs, and undergoes a protease-mediated conversion from a ribonuclease to a deoxyribonuclease.

PMID: 20223951

"Our findings that C. elegans DCR-1 is involved in generating TUNEL-reactive DNA breaks in apoptotic cells that are later resolved by downstream apoptotic nucleases such as CPS-6 and NUC-1 and that DCR-1 promotes, and even is required for, apoptosis in sensitized genetic backgrounds reveal an unexpected role of DCR-1 in apoptosis and in initiating apoptotic DNA degradation."

"The proteolytic mechanism we discovered, through which a ribonuclease is disabled and converted into a DNase, may have more general implications on regulation of RNases and DNases, RNA and DNA binding proteins, and their associated cellular functions."

As stated in the F1000 review: "It is of interest to see 1) if this interplay between the small RNA processing pathway and the apoptosis pathway is conserved in mammals and 2) if this cleavage-mediated substrate change can be found in other nucleases."

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All things below are purely based on my memory and limited reading of the focal adhesion field. If any evidence against it, please let me know :-) If you want to test my model, feel free; because I am not studying in the field of focal adhesion dynamics.

First, let me define "retrograde flow". "The flow of fluid in a direction other than normal, as in regurgitation.". It is widely used in the actin filed, as show in the diagram below. But, as far as I know, this concept has never been introduced into focal adhesions, because focal adhesions are anchored structure - how do they move, even retrograde flow? I would say that if focal adhesion proteins move "backwards" I define focal adhesion flows retrograde.

The assemble of focal adhesion has been extensively studied. With the help of super-high resolution microscopy, especially PALM, the molecular model and assemble dynamics of focal adhesions have been clearly demonstrated. However, how focal adhesions disassembled/remodeled has not been convincingly and clearly presented from the molecular level. What I am proposing here is an alternative way for focal adhesions to disassemble. Considering the normal way for focal adhesions disassembly is quite efficient and focal adhesions disassemble whenever they are no longer needed, like the left column of the following diagram.

In the alternative (right column above), inefficient focal adhesion disassembly, happens when the normal/efficient way is inhibited/disconnected/disrupted/altered - demonstrated as focal adhesion retrograde flow:

• Only happens in the disassembly phase, not the assembly phase of focal adhesion.
• It is inefficient, mainly in the recycling of focal adhesion proteins. Thus, more functional focal adhesion proteins are required to exert "normal" cellular events.
• It is bound to actin filaments, which is the driving force of retrograde flow. Any treatment disrupts actin retrograde flow should be able to disrupt focal adhesion retrograde flow. Also, any treatment disconnect the focal adhesion from the actin cytoskeleton should be able to disrupt focal adhesion retrograde flow.
• It should be possible to generate kymograph of focal adhesion retrograde flow, which can be used to study the correlation with actin retrograde flow.
• Treatments affect the stability of focal adhesions might affect the focal adhesion retrograde flow.
• The focal adhesion retrograde flow "slowly" drains the focal adhesion and causes the "shrinkage" of the size of focal adhesion.
• After photo-bleaching correction, the total amount of fluorescence from the focal adhesion retrograde flow track should stay the same during the time of retrograde flow, although the track is elongating due to the flow.
• Most of, if not all, focal adhesion proteins should be observed in the retrograde flow with similar dynamics. The ECM binding proteins should not be found in the focal adhesion retrograde flow.

Below is a diagram about how people currently think the actin/myosin network connected with the focal adhesions (this is the only reference I checked while typing this post). It is highly possible that in certain condition, some focal adhesion protein will be tightly bound to actin filaments and fades as retrograde flow ...

Science. 2007 Jan 5;315(5808):111-5.

Differential transmission of actin motion within focal adhesions.

Hu K, Ji L, Applegate KT, Danuser G, Waterman-Storer CM.
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Cell migration requires the transmission of motion generated in the actin cytoskeleton to the extracellular environment through a complex assembly of proteins in focal adhesions. We developed correlational fluorescent speckle microscopy to measure the coupling of focal-adhesion proteins to actin filaments. Different classes of focal-adhesion structural and regulatory molecules exhibited varying degrees of correlated motions with actin filaments, indicating hierarchical transmission of actin motion through focal adhesions. Interactions between vinculin, talin, and actin filaments appear to constitute a slippage interface between the cytoskeleton and integrins, generating a molecular clutch that is regulated during the morphodynamic transitions of cell migration.

PMID: 17204653

Posted 2010-3-29 by Liang C.

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