Professor Mary McCaffrey
About the Molecular Cell Biology (MCB) Research Group
Our research focuses on vesicular trafficking events controlled by the monomeric Rab GTPases. Rab proteins are master controllers of eukaryotic intracellular trafficking events of both the secretory and endocytic pathway. Intracellular trafficking underpins a wide range of crucial cell physiological processes such as secretion and uptake of materials into cells, cell division/cytokinesis, cell migration, insulin response events and memory acquisition processes. Thus, clarifying molecular and cellular events of vesicular trafficking is crucial not only to the understanding of healthy cellular processes, but also to decipher the basis of many major diseases ranging from cancer metastasis, neurodegeneration, and diabetes.
Our research concentrates on Rab GTPases that control the endocytic recycling pathway (Rab4, Rab11, and Rab14) and in particular the molecular and cellular characterisation of:
- The Rab11-FIPs – a family of related effector proteins that were originally identified by their ability to interact with Rab11. We have since found that they can also interact with a number of other Rabs.
- Motor proteins that interact with Rab GTPases of the endocytic recycling pathway.
- Other proteins that modulate the activity of endocytic Rabs e.g. GRAB.
The methodologies that we employ include: molecular biology, protein:protein interaction technologies, tissue culture, immunofluorescence/confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET), biochemical assays, immunoblotting and immunoprecipitation assays.
Current Research Projects in the MCB Research Group
- RAB14 and the RAB11-FIPS
- RAB Interactions and Functional Control of the Motor Proteins Dynein and Myosin V
- Characterisation Of The GRAB Protein In The Context Of The RAB11
1. RAB14 and the RAB11-FIPS
Rab11 and Rab14 are two related Rab GTPases that are believed to function in endosomal recycling and Golgi/endosome transport processes. We, and others, have identified a group of proteins that interact with Rab11 and function as Rab11 effectors, known as the Rab11-FIPs (family of interacting proteins). This protein family has been sub-classified into two groups - class I FIPs [FIP2, RCP (Rab coupling protein) and Rip11 (Rab11-interacting protein)] and class II FIPs (FIP3 and FIP4). We have identified the class I FIPs as dual Rab-binding proteins by demonstrating that they also interact with Rab14 in a GTP-dependent manner. We have shown that these interactions are specific for the class I FIPs and that they occur via their C-terminal regions, which encompass the previously described RBD (Rab11-binding domain). Furthermore, we have shown that Rab14 significantly co-localizes with the TfnR (transferrin receptor), Rab11a and with the class I FIPs on the ERC (endosomal recycling compartment) during interphase.
References
Class I Rab11-family interacting proteins are binding targets for the Rab14 GTPase. Kelly EE, Horgan CP, Adams C, Patzer TM, Ní Shúilleabháin DM, Norman JC, McCaffrey MW. Biology of the Cell. 2009 Oct 12;102(1):51-62.
The C2 domains of the class I Rab11 family of interacting proteins target recycling vesicles to the plasma membrane. Lindsay AJ, McCaffrey MW. Journal of Cell Science. 2004 Sep 1;117(Pt 19):4365-75.
Characterisation of the Rab binding properties of Rab coupling protein (RCP) by site-directed mutagenesis. Lindsay AJ, McCaffrey MW. FEBS Letters. 2004 Jul 30;571(1-3):86-92.
Rab11-FIP2 functions in transferring recycling and associates with endosomal membranes via its COOH-terminal domain. Lindsay AJ, McCaffrey MW. The Journal of Biological Chemistry. 2002 Jul 26;277(30):27193-9.
Rab coupling protein (RCP), a novel Rab4 and Rab11 effector protein. Lindsay AJ, Hendrick AG, Cantalupo G, Senic-Matuglia F, Goud B, Bucci C, McCaffrey MW. The Journal of Biological Chemistry. 2002 Apr 5;277(14):12190-9.
2. RAB Interactions and Functional Control of the Motor Proteins Dynein and Myosin V
Dynein
Several protein families control intracellular transport processes in eukaryotic cells. We have shown that the Rab11 GTPase effector protein Rab11-FIP3 directly interacts with the dynein light intermediate chain 1 (DLIC-1) subunit of the cytoplasmic dynein 1 motor protein complex. Rab11a, FIP3 and DLIC-1 form a ternary complex and these proteins colocalise with each other in cells. We have demonstrated that association between FIP3 and DLIC-1 at the cell periphery precedes minus-end-directed microtubule-based transport, that FIP3 recruits DLIC-1 onto membranes, and that knockdown of DLIC-1 inhibits pericentrosomal accumulation of key endosomal-recycling compartment (ERC) proteins. In addition, we have demonstrated that expression of a DLIC-1-binding truncation mutant of FIP3 disrupts the ability of ERC proteins to accumulate pericentrosomally. On the basis of these and other data, we propose that FIP3 links the Rab11 GTPase and cytoplasmic dynein in order to mediate the transport of material from peripheral sorting endosomes to the centrally located ERC.
References
Dynein LIC1 localizes to the mitotic spindle and midbody and LIC2 localizes to spindle poles during cell division. Horgan CP, Hanscom SR, McCaffrey MW. Cell Biology International. 2010 Dec 14;35(2):171-8.
Rab11-FIP3 binds dynein light intermediate chain 2 and its overexpression fragments the Golgi complex. Horgan CP, Hanscom SR, Jolly RS, Futter CE, McCaffrey MW. Biochemical and Biophysical Research Communications. 2010 Apr 2;394(2):387-92.
Rab11-FIP3 links the Rab11 GTPase and cytoplasmic dynein to mediate transport to the endosomal-recycling compartment. Horgan CP, Hanscom SR, Jolly RS, Futter CE, McCaffrey MW. Journal of Cell Science. 2010 Jan 15;123(Pt 2):181-91.
Myosin V
Class V myosins are a family of actin-based motor proteins. Individuals that lack members of this family can suffer from rare autosomal recessive diseases such as Griscelli’s syndrome type 1 (GS1) or microvillus inclusion disease (MVID). We have demonstrated that myosin Va plays an important role in the formation of P bodies (small cytoplasmic foci involved in the storage and turnover of mRNA transcripts), and in the transport of an RNA binding protein called FMRP to the periphery of the cell. We have also found that myosin Va interacts directly with members of six different Rab subfamilies.
References
Myosin Va is Required for the Transport of Fragile X Mental Retardation Protein (FMRP) Granules. Lindsay AJ, McCaffrey MW. Biol Cell. 2013 Oct 31.
Identification and characterization of multiple novel Rab-myosin Va interactions. Lindsay AJ, Jollivet F, Horgan CP, Khan AR, Raposo G, McCaffrey MW, Goud B. Mol Biol Cell. 2013 Nov;24(21):3420-34.
Roles for myosin Va in RNA transport and turnover. McCaffrey MW, Lindsay AJ. Biochemical Society Transactions. 2012 Dec 1;40(6):1416-20.
Myosin Va is required for p body but not stress granule formation. Lindsay AJ, McCaffrey MW. The Journal of Biological Chemistry. 2011 Apr 1;286(13):11519-28
3. Characterisation Of The GRAB Protein In The Context Of The RAB11
Co-ordination of Rab GTPase function has emerged as a crucial mechanism in the control of intracellular trafficking processes in eukaryotic cells. We have found that GRAB/Rab3IL1 [guanine nucleotide exchange factor for Rab3A; RAB3A interacting protein (rabin3)-like 1], a protein that has previously be shown to act as a GEF (guanine nucleotide exchange factor) for Rab3a, Rab8a and Rab8b, is also a binding partner for Rab11a and Rab11b, but not the closely related Rab25 GTPase. We have demonstrated that exogenous expression of Rab11a and Rab11b shift GRAB's distribution from the cytoplasm onto membranes. The Rab11a/Rab11b-binding region of GRAB lies within its carboxy-terminus, a region distinct from its GEF domain and Rab3a-binding region. Finally, we have described a GRAB deletion mutant (GRABΔ223-228) that is deficient in Rab11-binding ability. These data identify GRAB as a dual Rab-binding protein that could potentially link Rab3 and Rab11 and/or Rab8 and Rab11-mediated intracellular trafficking processes. We are currently investigating the whether Rab11, GRAB, and Rab3 or Rab8 function together in a Rab cascade.
References
GRAB is a binding partner for the Rab11a and Rab11b GTPases. Horgan CP, Hanscom SR, McCaffrey MW. Biochem Biophys Res Commun. 2013 Nov 8;441(1):214-9.
