Biomedical subjects

Brian Storrie

Publications and source records attributed to Brian Storrie.

10 recordsLinked to original sources

PubMed2006

Capacity of the Golgi apparatus for cargo transport prior to complete assembly.

In yeast, particular emphasis has been given to endoplasmic reticulum (ER)-derived, cisternal maturation models of Golgi assembly while in mammalian cells more emphasis has been given to golgins as a potentially stable assembly framework. In the case of de novo Golgi formation from the ER after brefeldin A/H89 washout in HeLa cells, we found that scattered, golgin-enriched, structures formed early and contained golgins including giantin, ranging across the entire cis to trans spectrum of the Golgi apparatus. These structures were incompetent in VSV-G cargo transport. Second, we compared Golgi competence in cargo transport to the kinetics of addition of various glycosyltransferases and glycosidases into nascent, golgin-enriched structures after drug washout. Enzyme accumulation was sequential with trans and then medial glycosyltransferases/glycosidases found in the scattered, nascent Golgi. Involvement in cargo transport preceded full accumulation of enzymes or GPP130 into nascent Golgi. Third, during mitosis, we found that the formation of a golgin-positive acceptor compartment in early telophase preceded the accumulation of a Golgi glycosyltransferase in nascent Golgi structures. We conclude that during mammalian Golgi assembly components fit into a dynamic, first-formed, multigolgin-enriched framework that is initially cargo transport incompetent. Resumption of cargo transport precedes full Golgi assembly.

Biological Transport↗

PubMed2005

Cisternal rab proteins regulate Golgi apparatus redistribution in response to hypotonic stress.

We show that a physiological role of the extensively studied cisternal Golgi rab protein, rab6, is modulation of Golgi apparatus response to stress. Taking exposure of cells to hypotonic media as the best-known example of mammalian Golgi stress response, we found that hypotonic-induced tubule extension from the Golgi apparatus was sensitive to GDP-rab6a expression. Similarly, we found that Golgi tubulation induced by brefeldin A, a known microtubule-dependent process, was inhibited by GDP-restricted rab6a, rab6a', and rab33b, the most commonly studied cisternal rab proteins. These GDP-rab levels were sufficient to inhibit rab-induced redistribution of Golgi glycosyltransferases into the endoplasmic reticulum (ER), also a microtubule-dependent process, and to depress Golgi membrane association of the GTP-conformer of rab6. Nocodazole-induced Golgi scattering, a microtubule-independent process, also was inhibited by GDP-rab6a expression. In comparison, we found similar GDP-rab expression levels had little inhibitory effect on another microtubule-independent process, constitutive recycling of Golgi resident proteins to the ER. We conclude that Golgi cisternal rabs, and in particular rab6a, are regulators of the Golgi response to stress and presumably the molecular targets of stress-activated signaling pathway(s). Moreover, we conclude that rab6a can regulate select microtubule-independent processes as well as microtubule-dependent processes.

Base Sequence↗

PubMed2005

Maintenance of Golgi apparatus structure in the face of continuous protein recycling to the endoplasmic reticulum: making ends meet.

I focus here on the Golgi apparatus and the dynamic relationship between the Golgi apparatus, the central organelle of the secretory pathway, and the endoplasmic reticulum (ER). The proteins and lipids of the Golgi apparatus originate in the ER, and cargo proteins and lipids that also originate in the ER are processed and sorted within the Golgi apparatus. The Golgi apparatus is indeed the central organelle of the secretory pathway. Surprisingly, many, if not all, of the proteins and accompanying lipids of the Golgi apparatus cycle continuously between the Golgi and the ER. Neither the Cisternal Maturation nor the Vesicular Transport/Stable Compartment model of Golgi apparatus function predicts continuous cycling of Golgi resident proteins through the ER. Evidence for this cycling comes from multiple experimental approaches, including ER-exit block-revealed accumulation of recycled Golgi resident proteins in the ER, evidence for exchange of green fluorescent protein (GFP)-tagged Golgi proteins or their analogues between Golgi and ER pools, and cisternal rab overexpression-induced redistribution of Golgi resident proteins to the ER. The implications of Golgi protein cycling for the maintenance of Golgi structure in the interphase mammalian cell are discussed. The challenge for the future is to put Golgi resident protein cycling pathway(s) to protein machinery and to characterize the cumulative, weak, dynamic interactions that hold the Golgi apparatus together. In doing so, new paradigms of organelle biogenesis will emerge.

Animals↗

PubMed2005

Microinjection as a tool to explore small GTPase function.

Microinjection overcomes the plasma membrane barrier to the introduction of charged or large nonlipid soluble molecules into cells by the direct insertion of a hollow capillary micropipette into the cell. With the application of pressure, aqueous solution is then directly transferred into either the cytosol or the nucleus. I give specific examples of the application of this approach to the functional study of small GTPases of the Sar1, ARF, and rab family in membrane trafficking between the Golgi apparatus and endoplasmic reticulum (ER). The principles illustrated by these examples should be generally applicable to other small GTPases. Detailed protocols for capillary microinjection using semiautomated equipment are given.

Animals↗

PubMed2005

The steady-state distribution of glycosyltransferases between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10.

Several lines of evidence support a novel model for Golgi protein residency in which these proteins cycle between the Golgi apparatus and the endoplasmic reticulum (ER). However, to preserve the functional distinction between the two organelles, this pool of ER-resident Golgi enzymes must be small. We quantified the distribution for two Golgi glycosyltransferases in HeLa cells to test this prediction. We reasoned that best-practice, quantitative solutions would come from treating images as data arrays rather than pictures. Using deconvolution and computer calculated organellar boundaries, the Golgi fraction for both endogenous beta1,4-galactosyltransferase and UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase 2 fused with green fluorescent protein (GFP) was 91% by fluorescence microscopy. Immunogold labeling followed by electron microscopy and model analysis yielded a similar value. Values reflect steady-state conditions, as inclusion of a protein synthesis inhibitor had no effect. These data strongly suggest that the fluorescence of a GFP chimera with an organellar protein can be a valid indicator of protein distribution and more generally that fluorescent microscopy can provide a valid, rapid approach for protein quantification. In conclusion, we find the ER pool of cycling Golgi glycosyltransferases is small and approximately 1/100 the concentration found in the Golgi apparatus.

Endoplasmic Reticulum↗

PubMed2004

A method for the purification of Shiga-like toxin 1 subunit B using a commercially available galabiose-agarose resin.

We describe a procedure for the affinity purification of Shiga toxin 1 subunit B (SLTB) using a commercial galabiose-agarose resin. Recombinant SLTB was purified to 99% homogeneity in a single-step protocol, from the periplasmic extracts of Vibrio cholerae 0395 N1/pSBC54. SDS-PAGE of the affinity purified SLTB showed one band of 8 kDa MW. SLTB purified by this procedure retained its chemical and biological activity as demonstrated by re-binding to the galabiose-agarose resin, and receptor-mediated binding and uptake in Vero cells. The galabiose-agarose resin could isolate roughly 1mg of SLTB/mL of gel. The resin was stable over 3 years and 500 cycles/year of usage. Hence, this method is a straightforward approach to the large-scale preparation of SLTB at a reasonable cost.

Animals↗

PubMed2004

Dynamic nucleation of Golgi apparatus assembly from the endoplasmic reticulum in interphase hela cells.

Models of Golgi apparatus biogenesis and maintenance are focused on two possibilities: one is self-assembly from the endoplasmic reticulum, and the other is nucleation by a stable template. Here, we asked in three different experimental situations whether assembly of the Golgi apparatus might be dynamically nucleated. During microtubule depolymerization, the integral membrane protein p27 and the peripheral Golgi protein GM130, appeared in newly formed, scattered Golgi elements before three different Golgi apparatus cisternal enzymes, whereas GRASP55, a medial peripheral Golgi protein, showed, if anything, a tendency to accumulate in scattered Golgi elements later than a cisternal enzyme. During Golgi formation after brefeldin A washout, endoplasmic reticulum exit of Golgi resident enzymes could be completely separated from that of p27 and GM130. p27 and GM130 accumulation was onto newly organized perinuclear structures, not brefeldin A remnants, and preceded that of a cisternal enzyme. Reassembly was completely sensitive to guanosine 5'-diphosphate-restricted Sar1p. When cells were microinjected with Sar1pWT DNA to reverse a guanosine 5'-diphosphate-restricted Sar1p endoplasmic reticulum-exit block phenotype, GM130 and p27 collected perinuclearly with little to no exit of a cisternal enzyme from the endoplasmic reticulum. The overall data strongly indicate that the assembly of the Golgi apparatus can be nucleated dynamically by GM130/p27 associated structures. We define dynamic nucleation as the first step in a staged organelle assembly process in which new component association forms a microscopically visible structure onto which other components add later, e.g. Golgi cisternae.

Antineoplastic Agents↗

PubMed2003

Persistence of Golgi matrix distribution exhibits the same dependence on Sar1p activity as a Golgi glycosyltransferase.

We investigated the relative distributional persistence of Golgi "matrix" proteins and glycosyltransferases to an endoplasmic reticulum exit block induced by expression of a GDP-restricted Sar1p. HeLa cells were microinjected with plasmid encoding the GDP-restricted mutant (T39N) of Sar1p to block endoplasmic reticulum exit and then scored for the distribution of GM130 (Golgi matrix protein of 130 kDa), a cis located golgin; p27, a member of the p24 family of proteins; giantin, a protein that interacts indirectly with GM130; and the Golgi glycosyltransferase, N-acetylgalactosaminyltransferase-2 (GalNAcT2). All of these proteins lost their compact, juxtanuclear distribution and displayed characteristics of endoplasmic reticulum/cytoplasmic accumulation with the same dependence on plasmid concentration. The kinetics of redistribution of GM130 and GalNAcT2 were identical. Expression of Sar1pT39N displaced the COPII coat protein Sec13p from endoplasmic reticulum exit sites consistent with disruption of these sites. This occurred without disturbing the overall distribution of endoplasmic reticulum membrane. Furthermore, the reassembly of a juxtanuclear Golgi matrix as assayed by the distribution of GM130 following washout of the Golgi disrupting drug, brefeldin A, was blocked by microinjected Sar1pT39N plasmids. We conclude that the persistence, i.e. stability and maintenance, of Golgi matrix distribution and its reassembly following drug disruption are exquisitely dependent on Sar1p activity.

Autoantigens↗

PubMed2002

The Golgi apparatus: balancing new with old.

Most models put forward to explain cellular processes do not stand the test of time. The 'lucky' few that are able to survive extensive experimental tests and peer critique may eventually become dogmas or paradigms. When this happens, the amount of experimental data required to overturn the paradigm is extensive. To some, such inertia may seem prohibitive to scientific progress but rather, in our opinion, this helps to maintain a degree of coherence. It is needed so that experiments and interpretations may be conducted within relatively safe boundaries. In the field of protein transport in the secretory pathway, we have enjoyed a relatively stable and productive period for quite some time (more than 30 years!). It is only very recently that the field has entered into a phase where all bets seem to be off. As in any paradigm shift, the accumulation of experimental observations inconsistent with the old dogma eventually reached a critical point. As we 'reluctantly' dispense with the long-standing paradigm of forward vesicular transport, we face a time that is bound to be trying as well as exciting.

Animals↗

PubMedDate not supplied

Shiga-like toxin subunit B (SLTB)-enhanced delivery of chlorin e6 (Ce6) improves cell killing.

We used Shiga-like toxin B subunit (SLTB) to deliver the photosensitizer, chlorin e6 (Ce6), to Vero cells expressing the Gb3 receptor. Our aim was to provide an example of carrier-enhanced photodynamic cell killing with which to start a systematic consideration of photosensitizer delivery at the subcellular level. SLTB, in contrast to many other potential protein carriers, is delivered intracellularly to the Golgi apparatus and endoplasmic reticulum (ER). Ce6 was chosen both for its phototoxic properties and its potential for covalent conjugation with SLTB. Ce6-SLTB after cleanup contained < or =10% noncovalently bound Ce6. The noncovalent binding of porphyrins and chlorins to protein conjugates has been well documented, and hence the effective cleanup procedure is a significant accomplishment. We demonstrate that Ce6-SLTB enhances delivery of Ce6 to target cells as compared to free Ce6. In Vero cells, Ce6-SLTB was over an order of magnitude more photodynamically toxic than free Ce6. Moreover, we show that in the case of Ce6-SLTB, photosensitizer accumulation is in a combination of subcellular sites including mitochondria, Golgi apparatus, ER and plasma membrane. The occurrence in nature of diverse B subunit binding sites and the possibilities of varied intracellular delivery make optimized use of B subunit carriers attractive.

Animals↗