What does the Cullen Lab study?
Summary
For over twenty years the Cullen laboratory has been identifying the mechanistic basis by which proteins and lipids are sorted through a specific aspect of the human cell's intracellular membraneous maze, the endosomal network. We have studied a number of ancient, highly conserved protein complexes that act as molecular machines to orchestrate protein and lipid transport through this network.
Keep reading to find out more about the endosomal system and our research interests!
The Cullen Lab (+ rotation students). Christmas dinner 2023 @ Beerd Bristol.
Figure 1. The plasma membrane proteome is essential for cell function. (Kindly provided by Rebeka Butkovič).
Figure 2. Sequence dependent cargo sorting is highly orchestrated. (Kindly provided by Dr Emma Jones).
What is the endosomal system?
Maintenance of the plasma membrane proteome is essential for many cellular functions, including cell signalling, migration and nutrient uptake (figure 1). Dynamic turnover of cell surface proteins (termed ‘cargo’) is governed by the removal of proteins through endocytosis and repopulation from the biosynthetic pathway and endosomal recycling.
Endocytosed cargo enter vesicular structures called endosomes. Endosomes are part of the endolysosomal network, with cargo residing in this pathway destined for degradation in the lysosome. Alternatively, cargo can be returned to the cell surface or the trans-Golgi network via sequence dependent cargo sorting. Sequence dependent cargo sorting is highly orchestrated by cargo sorting machinery (figure 2). Perturbations to this system manifest in a multitude of diseases, such as neurodegeneration.
We are interested in how endosomal sorting machinery orchestrate sequence dependent cargo sorting. We combine a suite of biochemical, molecular and cellular approaches to delineate mechanistic details underpinning cargo sorting mechanisms.
SNX3/SNX27-Retromer
Retromer and sorting nexins (SNXs), in particular SNX3 and SNX27, facilitate cargo recognition and egress from endosomes. The Retromer complex is implicated in having a role in safeguarding neuronal function and synaptic integrity, with reduced brain levels associated with neurodegeneration, such as Alzheimer’s disease. Furthermore, endosomal swelling cytopathology is a bona fide early hallmark of neurodegenerative diseases (figure 3). Taken together, this indicates functional endosomal biology is central to neuronal function.
Our research questions are centred around SNX3/SNX27-Retromer’s (figure 4) role in neuronal biology. Utilising immortalised and primary cell cultures, we aim to dissect endosomal trafficking mechanisms relevant to disease.
Figure 3. Endosomal enlargement is an early hallmark of neurodegenerative diseases.
(Kindly provided by Kincaid Ingram).
Figure 4. (Left) SNX27- and (Right) SNX3-Retromer.
(Kindly provided by Rebeka Butkovič).
Figure 5. SNX17 enriches cargo at the endosomal retrieval subdomain. (Butkovič et al. 2024).
Figure 6. The Commander Complex. (Healy MD, McNally KE, Butkovič R, Chilton M et al. 2024).
SNX17 and the Commander Complex
The Commander trafficking pathway is essential for the intracellular sorting of distinct integral membrane proteins. Understanding of the Commander trafficking pathway and how perturbations drive disease pathogenesis are of central importance to the lab.
Since our discovery of SNX17-Retriever in 2017, we have focussed on developing our mechanistic understanding of the complex’s role at the endosome retrieval subdomain. Recent work has uncovered a process by which SNX17 autoregulates its ability to recognise cargo (figure 5).
Utilising recent advances in AlphaFold modelling, we were the first to identify and validate a structure for the Commander complex. The Commander comprises 16 subunits, arranged in two core sub-assemblies: the Retriever and CCC complexes. Retriever (VPS26C, VPS29, VPS35L) shares distant homology with Retromer. The CCC complex contains 12 components, CCDC22 (coiled coil domain-containing) and CCDC93 and ten COMMD (copper metabolism MURR1 [Mouse U2af1-rs1 region 1] domain) family members COMMD1-COMMD10. The last component is DENND10 (differentially expressed in normal and neoplastic cells-containing protein 10, also called FAM45A) (figure 6).
Our research questions are centred around the Commander complex's role at the endosomal sorting subdomain and how relevant disease mutations perturb this function.
ESCPE Complexes
Cargo egress requires tubulovesicular carrier formation at the endosomal retrieval subdomain. The evolutionary conserved endosomal SNX-BAR proteins achieve this using the ridge BAR domains that enforce their curve structure onto membranes by interacting with themselves, shallow ordered insertions of amphipathic helices and interacting electrostatically with the membrane (figure 7).
ESCPE-1 is a complex form by SNX-BAR proteins. In addition to tubulovesicular carrier biogenesis, ESCPE-1 directly recognizes cargo and promotes its recycling. Its cargo has impact in lysosome degradation (CI-MPR), cell growth and cancer (IGF1R) and protein trafficking and SARS-COV-II infection (NRP1). It also promotes SNX27-Retromer indirect cargo recycling via its interaction with SNX27. We are also interested in other SNX-BAR proteins as SNX4 and SNX8 (figure 8).
Our research is focused on dissecting molecular detail of how ESCPE complexes mediate endosomal recycling. By leveraging in silico, in vitro and in cellulo techniques we aim to establish their role at the retrieval subdomain.
Figure 7. SNX-BAR Oligomeric Assembly on Tubular Structures. (Simonetti B, Daly JL and Cullen PJ 2023).
Figure 8. Endosomal SNX-BAR Complexes. (Simonetti B, Daly JL and Cullen PJ 2023).