Calcium phosphate transfection protocol

Protocol for calcium phosphate transfection of mammalian cells with siRNA.

This is derived from the protocol of Chen and Okayama, 1987.

Originally “told to us” by Harry Mellor (https://mellorlab.wordpress.com/). It works very well. As Chen and Okayama themselves point out, “the crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation”.

siRNA by calcium phosphate

Day 1

Trypsinize and count cells
Seed cells at 75000 cells/ml in DMEM with 10% FCS.

This does depend on cell type, growth rate etc.

Day 2

For a 10cm (10ml) plate prepare:
10 µl siRNA (20µM stock) in 500µl filter-sterilised 0.25 M CaCl2
Add 500µl filter-sterilised BBS (50 mM BES, pH 6.95, 280 mM NaCl, 1.5 mM Na2HPO4).
Mix by pipetting gently and leave at room temperature for 15-20 min
Add siRNA transfection mix dropwise to plate whilst swirling
Remove plate to a 3% CO2 incubator.

Day 3

Wash plate once with PBS then add fresh medium and return to 5% CO2 incubator

Day 4 or 5 depending on application

Harvest/do experiment

 

We usually leave cells for 72h for effective depletion but this is target dependent.

Works well with a variety of cells including HeLa and RPE1.

Comments on “Myosin-va and dynamic actin oppose microtubules to drive long-range organelle transport”

I have evaluated the following article for F1000 Prime:

Myosin-va and dynamic actin oppose microtubules to drive long-range organelle transport.
RD Evans, C Robinson, DA Briggs, DJ Tooth, JS Ramalho, M Cantero, L Montoliu, S Patel, EV Sviderskaya and AN Hume
Curr Biol 2014 Aug 4; 24(15):1743-50  DOI: 10.1016/j.cub.2014.06.019

These comments were originally posted at: http://f1000.com/prime/718509369

This is a fascinating paper that reports the perhaps unexpected finding that the long-range centrifugal transport of melanosomes is driven by myosin rather than kinesin motors. Considerable previous work in this area has demonstrated clearly defined roles for cytoplasmic dynein and kinesin-2 in bidirectional motility of melanosomes along microtubules. Here, the authors use unbiased monitoring of organelle positions, made possible through the use of constrained patterning, combined with some clever use of myosin mutants. The authors make some interesting points in the discussion about the potential physiological relevance of this mechanism in driving the dispersal of pigment granules between cells. From a more fundamental, mechanistic perspective, I would be fascinated to see the outcome of similar experiments in which the kinesin-2 motor was also perturbed. Clearly, there is much more to be uncovered in melanosome biology, notably in terms of the relative contributions of the different motor/filament systems to the maturation and dispersal of pigment granules.

Recommended Article (F1000 Prime) cTAGE5 and Sec12

I recommended the following article for F1000 Prime.

Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export.
K Saito, K Yamashiro, N Shimazu, T Tanabe, K Kontani and T Katada
J Cell Biol 2014 Sep 8 DOI: 10.1083/jcb.201312062

This work has some significant implications for the organization and mechanisms of secretory cargo export from the endoplasmic reticulum (ER). Assembly of the COPII complex that drives this event occurs at spatially restricted sites on the ER membrane known as ER exit sites (ERES). Here, Saito and colleagues show that cTAGE5, a protein shown previously to be required for collagen export, is required to localize Sec12 to ERES. This is important because Sec12 is the initiator of COPII assembly. Sec12 acts as the guanine nucleotide exchange factor for Sar1, which, in its GTP-bound state, recruits the rest of the COPII complex. Previous work {1} had shown that Sec12 was localized across the ER with possibly some minor concentration in ERES. Here, using newly developed antibodies, the authors show that Sec12 is highly enriched at ERES, dependent on the presence of cTAGE5. The authors then tie this to the role of cTAGE5 in driving collagen secretion, notably of collagen VII. Perhaps more significantly, this work could implicate cTAGE5, and by extension its partner TANGO1, as a more fundamental component of the COPII budding machinery. cTAGE5 does not appear to be absolutely required for ongoing transport of many cargoes; there does, however, appear to be a kinetic delay in the delivery of cargo from the ER to Golgi.
There are some other important features to the data: cTAGE5 doesn’t appear to be required for the localization of Sec16 to ERES, nor is collagen VII required to maintain cTAGE5 at ERES (which is consistent with roles beyond that in collagen VII secretion alone). cTAGE5 also appears to be interdependent with its known interacting partner, TANGO1, for much of its functions.
The conclusion is really that cTAGE5 bridges Sec12 and TANGO1. The key question remains how these interactions are controlled and integrated with other factors known to be required for COPII function, especially in the context of collagen secretion.

References:
{1} The mammalian guanine nucleotide exchange factor mSec12 is essential for activation of the Sar1 GTPase directing endoplasmic reticulum export. Weissman JT, Plutner H, Balch WE. Traffic 2001 Jul; 2(7):465-75 PMID: 11422940

This recommendation was originally posted here:  http://f1000.com/prime/718765169

F1000Prime eval: Dynein and SQSTM1

I recommended this article on F1000 Prime, comments appended below.

Interaction of SQSTM1 with the motor protein dynein: SQSTM1 is required for normal dynein function and trafficking.
L Calderilla-Barbosa, ML Seibenhener, Y Du, MT Diaz-Meco, J Moscat, J Yan, MW Wooten and MC Wooten
J Cell Sci 2014 Jul 11 DOI: 10.1242/jcs.152363

Here, Calderilla-Barbosa and colleagues define an interaction between the cytoplasmic dynein motor protein complex and the SQSTM1 protein (also known as p62) a key component of the machinery that removes aggregated proteins from the cell.

This work defines the binding site on SQSTM1 and is consistent with a direct interaction with the dynein intermediate chain subunit. The data suggest that the binding site for SQSTM1 on dynein could be shared with that of the histone deacetylase HDAC6. The complex interplay between these three components remains to be defined in any real depth. However, models are proposed within the paper where the dynamic interplay of HDAC6-mediated microtubule acetylation/deacetylation, SQSTM1-mediated binding of aggregated cargo, and dynein-based motility acts to control the removal of aggregated proteins from within cells.

While much of the data rely on immunofluorescence of dynein, which must always be treated with caution owing to the difficulty of detecting specific dynein immunolabelling in cells, it is clear from this work that dynein and SQSTM1 have an interdependent relationship in the removal of aggregated proteins from cells.

These comments were originally posted at:

http://f1000.com/prime/718489227

Other recent F1000 evaluations

I have not recently updated my blog to include my F1000 evaluations; I now include these missing evaluations here for the benefit of those who do not subscribe to F1000. All of the following were first published on F1000 Prime in the last 12 months.

The GTPase IFT27 is involved in both anterograde and retrograde intraflagellar transport.

Huet D, Blisnick T, Perrot S, Bastin P. elife 2014; 3:e02419 PMID: 24843028 DOI: 10.7554/eLife.02419.001

This fascinating paper exploits Trypanosomes as a model system to define a role for the intraflagellar transport (IFT) protein IFT27 in flagellar function. IFT27 is a known component of the IFT-B particle mediating transport from the flagellar base to tip (anterograde IFT); here, the authors show that the GTPase function of IFT27 is also required to load the retrograde IFT particle (IFT-A) and its associated motor (dynein-2) into cilia. As such, this work defines IFT27 as a critical control point not only for anterograde but also for retrograde IFT. A key point is that IFT27 is a small Rab-like GTPase, and the authors convincingly demonstrate a role for this enzyme activity in the transport process. In some ways this provides further analogy between IFT-mediated transport and canonical membrane trafficking steps in which Rab proteins have critical roles in controlling timing and directionality.

 

Proteins of the Ciliary Axoneme Are Found on Cytoplasmic Membrane Vesicles during Growth of Cilia.

Wood CR, Rosenbaum JL. Curr Biol 2014 May 19; 24(10):1114-20 PMID: 24814148 DOI: 10.1016/j.cub.2014.03.047

This paper provides good evidence that structural proteins of the ciliary axoneme are transported into cilia on vesicular structures. Using Chlamydomonas as a model system, the data show that axonemal components and intraflagellar transport (IFT) particle proteins are delivered to the base of cilia on the surface of vesicles. These then associate with transitional fibres at the point of contact with the cell membrane. The process is particularly evident during cilia formation when a burst of synthesis of the ciliary components must be matched by an upregulation of their delivery. The work suggests an important and ongoing association of axonemal proteins with membranes. This and other work also support the maintained association of IFT proteins with membranes during transport within the cilium. This is therefore a significant paper in that it provides an integrated model for the transport of both membrane and axonemal proteins into cilia.

 

SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium-triggered fusion.

Bharat TA, Malsam J, Hagen WJ, Scheutzow A, Söllner TH, Briggs JA. EMBO Rep 2014 Mar 1; 15(3):308-14 PMID: 24493260 DOI: 10.1002/embr.201337807

The paper provides strong supporting evidence in favour of a membrane deformation event early in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent fusion. I concur entirely with what has already been said by Liangyi Chen and Junmei Fan and especially by Robert Burgoyne in his evaluation. Providing a mechanistic basis for some early electron microscopy (EM) is a lovely way to close the loop.

 

An Unconventional Secretory Pathway Mediates the Cilia Targeting of Peripherin/rds.

Tian G, Ropelewski P, Nemet I, Lee R, Lodowski KH, Imanishi Y. J Neurosci 2014 Jan 15; 34(3):992-1006 PMID: 24431457 DOI: 10.1523/JNEUROSCI.3437-13.2014

This paper provides some further evidence for the somewhat controversial idea of a non-conventional secretion pathway operating from the early secretory pathway to the cilium. Evidence on this originally came from the Witzgall lab looking at the trafficking of polycystin-2 {1}. Here, the authors use cell culture models and Xenopus to show that Peripherin-2/rds seems to be trafficked in a similar manner. Key evidence for this comes from glycosylation assays monitoring sensitivity to endoglycosidase H (EndoH). Canonical trafficking of glycoproteins results in an acquisition of EndoH resistance as a result of sugar chain modification in the Golgi. While there is some potential for slightly different interpretations, the data are broadly consistent with the idea of a non-canonical pathway operating in the delivery of cargo to the cilium.

References

Polycystin-2 takes different routes to the somatic and ciliary plasma membrane.

Hoffmeister H, Babinger K, Gürster S, Cedzich A, Meese C, Schadendorf K, Osten L, de Vries U, Rascle A, Witzgall R. J Cell Biol 2011 Feb 21; 192(4):631-45 PMID: 21321097 DOI: 10.1083/jcb.201007050

 

A Regulator of Secretory Vesicle Size, Kelch-Like Protein 12, Facilitates the Secretion of Apolipoprotein B100 and Very-Low-Density Lipoproteins.

Butkinaree C, Guo L, Ramkhelawon B, Wanschel A, Brodsky JL, Moore KJ, Fisher EA. Arterioscler Thromb Vasc Biol 2013 PMID: 24334870 DOI: 10.1161/ATVBAHA.113.302728

This article is important in that it extends our understanding of the molecular mechanisms governing the formation of large COPII vesicles at the endoplasmic reticulum (ER). Previously, the Rape lab has shown that the ubiquitin conjugation cofactor Klhl12 is required for the COPII-dependent formation of procollagen containing carriers {1}, likely through ubiquitylation of the outer layer COPII subunit Sec31. Here, Fisher and colleagues show that another atypically large cargo, apolipoprotein particles, also require Klhl12. While the paper does not fully explore the similarities or potential differences in mechanisms, the data do support a role for Klhl12 more generally in the export of unusually large cargo from the ER.

References

Ubiquitin-dependent regulation of COPII coat size and function.

Jin L, Pahuja KB, Wickliffe KE, Gorur A, Baumgärtel C, Schekman R, Rape M. Nature 2012 Feb 23; 482(7386):495-500 PMID: 22358839 DOI: 10.1038/nature10822

 

Microtubules that form the stationary lattice of muscle fibers are dynamic and nucleated at Golgi elements.

Oddoux S, Zaal KJ, Tate V, Kenea A, Nandkeolyar SA, Reid E, Liu W, Ralston E. J Cell Biol 2013 Oct 28; 203(2):205-13 PMID: 24145165 DOI: 10.1083/jcb.201304063

This article shows that static Golgi elements in flexor digitorum brevis (FDB) muscle fibers organize the microtubule network. The surprising finding is that this is not through a Golgi-nucleated microtubule mechanism involving proteins such as AKAP450 and GCC185 but instead through centrosomal proteins pericentrin and gamma-tubulin.
These experiments stemmed from the observation of microtubule bundles in these cells where an apparently stable microtubule lattice was seen to be decorated with dynamic EB3 puncta along their length. EB3 is known to only decorate growing microtubule ends. This was then explained through the good use of super-resolution microscopy (in this case gated stimulated emission depletion [GSTED]). Here, Oddoux and colleagues showed the presence of microtubule bundles emanating from clearly definable hubs. These hubs turned out to be Golgi elements, nucleating new microtubules which then grew alongside pre-existing ones. These in vitro data are also supported by in vivo data from intravital imaging. These experiments convincingly demonstrate the relevance of these findings in vivo.

The data are very carefully quantified and present a compelling case. The story adds nicely to our understanding of the relationship between the Golgi and centrosomal proteins in an interesting and important model system. The authors are now also well placed to determine the relevance of this organization and these mechanisms to Duchenne muscular dystrophy where loss of microtubule organization is thought to underlie the disease pathology {1}.

References

Microtubules underlie dysfunction in duchenne muscular dystrophy.

Khairallah RJ, Shi G, Sbrana F, Prosser BL, Borroto C, Mazaitis MJ, Hoffman EP, Mahurkar A, Sachs F, Sun Y, Chen YW, Raiteri R, Lederer WJ, Dorsey SG, Ward CW. Sci Signal 2012; 5(236):ra56  PMID: 22871609 DOI: 10.1126/scisignal.2002829

 

Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division.

Paridaen JTML, Wilsch-Bräuninger M, Huttner WB. Cell 2013 Oct 10; 155(2):333-44 PMID: 24120134 DOI: 10.1016/j.cell.2013.08.060

This is one of those papers that has such a simple underpinning observation that one wonders how it has not been seen before. The authors identify an Arl13b-positive membrane that is endocytosed from the primary cilium as the cell enters mitosis. This associates selectively with the mother centriole and gives this a head-start in terms of ciliogenesis and therefore in the ability to respond to sonic hedgehog signals. The paper demonstrates this in a number of different ways, both in vitro and in vivo, and shows some evidence for the importance of this asymmetric ciliogenesis in stem cell function.
Many questions remain as a result of this work. The evidence here strongly supports a model where this internalized Arl13b-positive membrane acts as a primer for ciliogenesis. Not only will it be important to define how this selective association occurs but whether and how selective biosynthetic trafficking to this compartment reinforces this process to drive subsequent cilia formation on exit from mitosis. I strongly recommend a look at the accompanying mini-review from Hoerner and Stearns as well {1}.

References

Remembrance of cilia past. Hoerner C, Stearns T. Cell 2013 Oct 10; 155(2):271-3 PMID: 24120128 DOI: 10.1016/j.cell.2013.09.027

 

Cerebral organoids model human brain development and microcephaly.

Lancaster MA, Renner M, Martin CA, Wenzel D … Homfray T, Penninger JM, Jackson AP, Knoblich JA.  Nature 2013 Sep 19; 501(7467):373-9 PMID: 23995685 DOI: 10.1038/nature12517

This is an outstanding manuscript that demonstrates not only the development of a system to model the human brain in vitro but also then applies this technique to prove that it can model complex human brain diseases. The authors develop what they term ‘cerebral organoids’ through differentiation of human pluripotent stem cells in a 3D culture system. They also develop organoids from patient-derived stem cells to show that the observed microcephaly caused by mutation in CDK5RAP2 is recapitulated in this system. The microcephaly phenotype is also evident after short hairpin RNA (shRNA) knockdown of CDK5RAP2, further demonstrating the applicability of this method. This paper provides an outstanding example for teaching purposes and of course will likely be widely applied to human developmental brain disorders.

 

A conserved role for atlastin GTPases in regulating lipid droplet size.

Klemm RW, Norton JP, Cole RA, Li CS … Farese RV Jr, Blackstone C, Guo Y, Mak HY.  Cell Rep 2013 May 30; 3(5):1465-75 PMID: 23684613 DOI: 10.1016/j.celrep.2013.04.015

The concept that atlastin is important for lipid metabolism raises new questions about the way that mutations in this protein cause human disease. The paper also points to a key link between endoplasmic reticulum morphology and lipid droplet biogenesis, which could have important implications for many other studies in this field.

 

ER exit sites are physical and functional core autophagosome biogenesis components.

Graef M, Friedman JR, Graham C, Babu M, Nunnari J. Mol Biol Cell 2013 PMID: 23904270 DOI: 10.1091/mbc.E13-07-0381

This paper convincingly identifies a key role for endoplasmic reticulum (ER) exit sites (ERESs) in autophagosome formation in Saccharomyces cerevisiae. These data show that autophagosomes form in close proximity to these specialised invaginations of the ER where COPII vesicles assemble.

Using a broad-ranging forward proteomics approach, Graef et al. defined multiple protein-protein interactions between autophagy proteins and vesicle trafficking proteins, most notably those that comprise the COPII coat. Key interactions were defined between COPII and proteins of the PI3-kinase and Atg9 complexes that initiate autophagosome formation. It is notable that the Sec23 subunit of the COPII coat appears to be at the hub of this network of interactions. This subunit also interacts with other key cellular machineries directing multiple functions of COPII and ensuring a directional flow to traffic (see {1}). Fluorescence imaging showed that early autophagosome structures formed adjacent to ERESs. Consistent with a key role for ERESs in this process, inhibiting ERES function using a temperature-sensitive form of Sec12 inhibited autophagic flux.

The authors conclude that ERESs act in the mechanical process of ERS formation and suggest that one role could be to tether the nascent autophagosome as it expands. While it is not yet clear exactly how ERESs function in autophagosome formation, the work places ERESs as acting downstream of Atg1 kinase complex localisation and speculate that ERESs provide membrane, and/or somehow scaffold, autophagosome assembly.

Interestingly, the authors extended their findings to higher eukaryotic cells, showing similar co-localisation between autophagy components and ERESs (labelled with mCherry-Sec16B) in Cos-7 cells. Exactly how ERESs direct autophagosome assembly, the identity of the proposed ERES-tether complex, and how the autophagosome is eventually released from its association with the ER remain to be defined.

References

Coordination of COPII vesicle trafficking by Sec23. Fromme JC, Orci L, Schekman R. Trends Cell Biol 2008 Jul; 18(7):330-6 PMID: 18534853 DOI: 10.1016/j.tcb.2008.04.006

 

Comments on: Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase.

I provided some comments on this paper for F1000 Prime.

Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase.

Szyk A, Deaconescu AM, Spector J, Goodman B … Ziolkowska NE, Kormendi V, Grigorieff N, Roll-Mecak A.  (2014) Cell  157(6):1405-15 PMID: 24906155 DOI: 10.1016/j.cell.2014.03.061

This paper combines structural biology and biochemical assays to define a mechanism by which tubulin acetyl transferase (TAT) acts almost exclusively on stable microtubules. In some ways this is a very simple paper with clear and defined outcomes. X-ray crystallography shows that the catalytic site of TAT is not optimised to deprotonate the target lysine residue (Lys-40) within tubulin. Deprotonation is a prerequisite for acetylation and therefore this sub-optimal arrangement greatly decreases the catalytic rate. The authors then use some clever biochemical tricks to demonstrate the preference of TAT for assembled microtubules rather than monomeric or even dimeric tubulin. Lys-40 lies on the inside of assembled microtubule filaments, potentially posing an accessibility problem. A clever experiment was done to show that TAT does indeed enter the lumen of the microtubule. TAT conjugated to a large diameter (1 micron) bead was not capable of acetylating microtubules. This is the clearest evidence yet that acetylation occurs within the microtubule lumen. Live imaging of GFP-TAT showed that it diffuses freely within the lumen of microtubules and acts stochastically in acetylating the length of the assembled microtubule structure. Thus, entry and diffusion of TAT into the microtubule are not limiting factors in microtubule acetylation — that limit is defined by the slow catalytic rate of the TAT enzyme itself.

The work has broad-reaching implications since acetylated microtubules have been shown to be preferred by kinesin-1 motor {1} and that the microtubules that make up the axonemes of cilia and flagella are heavily acetylated {2}.

A nice commentary accompanies this article; it contains a nice model explaining the concepts and is also worth reading {3}.

References

1.
Microtubule acetylation promotes kinesin-1 binding and transport.

Reed NA, Cai D, Blasius TL, Jih GT, Meyhofer E, Gaertig J, Verhey KJ. Curr Biol 2006 Nov 7; 16(21):2166-72

2.
Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms.

Piperno G, Fuller MT. J Cell Biol 1985 Dec; 101(6):2085-94

PMID: 2415535
3.
A Slow Dance for Microtubule Acetylation.

Kull FJ, Sloboda RD. Cell 2014 Jun 5; 157(6):1255-1256

Comments on: Sec23 homolog Nel1 is a novel GTPase-activating protein for Sar1 but does not function as a subunit of the COPII coat

I recently evaluated this paper for F1000 Prime.

Sec23 homolog Nel1 is a novel GTPase-activating protein for Sar1 but does not function as a subunit of the COPII coat.

Kodera C, Yorimitsu T, Sato K. (2014) J Biol Chem PMID: 24947508 DOI: 10.1074/jbc.M114.553917

This paper suggests a possible mechanism by which the Sar1 GTP-binding protein is kept inactive outside of the context of the COPII coat. The work provides a relatively straightforward characterization of the Nel1 gene product in Saccharomyces cerevisiae. COPII vesicles bud from the endoplasmic reticulum (ER) to drive the first membrane trafficking step in the eukaryotic secretory pathway. The process is driven by activation of the small GTP-binding protein Sar1. A key feature of COPII assembly is spatial and temporal control. The most important findings are that it can act as a GTPase-activating protein for Sar1 and that it is not a component of the COPII coat. These data suggest that Nel1 could act to maintain Sar1 in an inactive state outside of the context of COPII vesicle budding. Whether Nel1 has roles (such as in signalling) outside of the context of COPII regulation remains to be defined.

This evaluation was originally published on Faculty of 1000 Prime.