Dr. Robinson's laboratory seeks to gain detailed knowledge of the mechanisms behind pathogenicity and disease through elucidating structures of key components involved in the progression of these disorders. The laboratory is interested in all areas of aberrant function and misregulation of proteins in conditions arising from genetic mutations or external challenges. One central, but not limiting, theme of the laboratory is the harnessing of force-generating polymerization machines in (mis)driving critical biological processes.

Bacterial Inheritance of Resistance and Toxicity
Many pathogenic bacteria encode their toxins and antibiotic resistance proteins on DNA plasmids. During cell division, large plasmids need to be actively segregated to ensure the faithful inheritance of these pathogenic agents. Actin-like proteins are frequently employed as the force providing motor to separate pairs of plasmids prior to cytokinesis. These actin-like polymerizing motors are required to be specific for their respective plasmids. In a multi-plasmid background, these segregating machineries have diversified their architectures and dynamics in order for the bacteria to reliably partition the full range of genetic material (Fig. 1). The laboratory uses structural and biophysical techniques to understand the mechanisms of inheritance of pathogenic traits by bacteria. Notable successes have been the elucidation of the structure and dynamics of segregation machineries from: 1) The multi-antibiotic resistance encoding R1 plasmid from Escherichia coli and Salmonella enterica; 2) The Staphylococcus aureus pSK41 plasmid that confers multiple resistance to several antibiotics, disinfectants and antiseptics in some forms of MRSA; and 3) The toxin-harboring plasmid pE88 from the tetanus causing bacteria, Clostridium tetani. We welcome contact from laboratories studying bacterial diseases and from bioengineers with a view to integrating these molecular motors into nanodevices.

Metastasis
A macrophage moving towards a site of infection and a developing (or regenerating) neuron extending towards its target tissue, are just two examples of normal cellular locomotion. These processes are driven through harnessing the force created by actin polymerization behind the advancing membrane. A host of actin-regulating proteins direct this process, controlling the spatial and temporal coordination of the polymerization to ensure concerted movement (Fig. 2). Mutation and/or misregulation of one or more of these regulating proteins are observed in many types of cancer. Often a change in the expression of polymerization-promoting regulators is observed at the switch between sedentary and invasive tumours. This laboratory studies the structural basis of actin regulation in order to understand the effects of mutations and misregulation on these regulators in metastasis. Current proteins of interest include: Adseverin, CapG, capping protein, gelsolin, thymosin-β4 and WH2-domain containing proteins. We encourage contact from other laboratories working on metastasis and actin regulation.

Actin Scavenging System
The preponderance of actin within cells poses a major challenge to the microcirculation in conditions of massive cell lysis, such as severe trauma and sepsis, which release the cell contents into the blood circulation. To overcome this, blood plasma contains an actin scavenging system comprised of two proteins, gelsolin and Gc-globulin. These two proteins dismantle actin filaments and remove actin monomers from the blood plasma (Fig. 3). In severe cases, this system can be overwhelmed resulting in circulating actin filaments and blockage of the microcirculation. This innate blood plasma actin scavenging system is acutely challenged in cases of massive cell death, such as severe trauma, burns and liver failure. The laboratory uses structural biology to understand the actin scavenging mechanisms with the aim of supplementing the system in critical conditions. We are keen to establish ties with clinicians and clinician scientists that are interested in conditions where extracellular actin may play a role in secondary injuries, such as: crushing injuries, sepsis, ARDS, MODS and cystic fibrosis.

Familial Amyloidosis (Finnish type)
One success of the laboratory has been in understanding the mechanism of the disease Familial Amyloidosis (Finnish type). FAF is a systemic polynueropathy that is dominantly inherited. Manifestations of the disease generally appear in middle age and include skin changes, as well as corneal lattice and cranial dystrophies. Single base changes in the gelsolin gene result in the production of the protein with mutation at Asp187. During transit through the trans-Golgi network, the mutant gelsolin transiently encounters furin, which hydrolyzes the peptide bond between Arg172 and Ala173. The resultant 68-kDa C-terminal fragment of gelsolin is secreted into the circulation and undergoes subsequent cleavage by β-gelsolinase to ultimately to produce an amyloidogenic 71-residue fragment of gelsolin, Ala173-Met243. The progression of FAF involves the assembly of this fragment into amyloid fibrils in the affected regions. This laboratory’s structural and biochemical data have identified Asp187 as a Ca2+-binding site, which mediates gelsolin’s conformational change required for activation (Fig. 4). Mutation of Asp187 abolishes the binding of the calcium ion, slowing the rate of activation and exposes the 172-173 peptide bond for cleavage by furin - the initiating step in the disease.

Techniques
The laboratory specializes in a range of structural biology and biophysical techniques, which include: protein crystallography, electron microscopy, Total Internal Reflection Fluorescence (TIRF) microscopy, Small Angle X-ray Scattering (SAXS), fluorescence, dynamic light scattering and SILAC for mass spectrometry (in collaboration with JG). These methods allow the determination of the conformations and dynamics of protein complexes and interactions, which provide understanding of how each protein performs, or fails to perform, its biological function. We are also actively engaged in drug design. The laboratory is heavily involved in method development: ranging from creating novel technologies to purify protein complexes from native sources; through designing advanced microfluidic cells for TIRF microscopy; to single molecule structure determination using the Free Electron Laser (FEL).

Collaborations, Students and Postdocs
The laboratory collaborates extensively within Singapore and internationally. Inter A-STAR Research Institute connections include joint projects with BII, ETC, ICES, IHPC, IMRE and the P53lab. The laboratory is keen to expand this portfolio. Similarly, we encourage applications from A*STAR Scholars, Returning Scholars and prospective Scholars, as well as from potential graduate students and postdocs that are either self financed or willing and eligible to apply for one of the following schemes:

A-STAR

EMBO

NTU

NUS