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Thymosin beta-4 (aliases: Thymosin β4, Tβ4, β-Thymosin, TMSB4X, FX, PTMB4, TB4X, TMSB4) is a protein that in humans is encoded by the TMSB4X gene (on the X-chromosome; there is also a suppressed Y-chromosome copy: TMSB4Y). Thymosin β4 has a molecular weight of 4921 g/mol and the protein consists of 43 amino acids:


Thymosin β4 is a member of the Beta thymosin family of proteins which have in common a sequence of about 40 amino acids (see: Beta Thymosins). Found almost exclusively in multicellular animals, Thymosin β4 was originally obtained from the thymus, along with in company with several other small proteins.

See also: Actin and Actin Dynamics

Relation to the WH2 sequence module

The N-terminal half of β-thymosins bears a strong similarity in amino acid sequence to a very widely distributed sequence module, the WH2 module. (Wasp Homology Domain 2 - the name is derived from Wiskott-Aldrich syndrome protein). Evidence from X-ray crystallography shows that this part of β-thymosins binds to actin in a near-identical manner to that of WH2 modules, both adopting as they bind, a conformation which has been referred to as the β-thymosin/WH2 fold. β-thymosins may therefore have evolved by addition of novel C-terminal sequence to an ancestral WH2 module. However, sequence similarity searches designed to identify present-day WH2 domains fail to recognise β-thymosins, (and vice versa) and the sequence and functional similarities may result from convergent evolution.

Biological activities of thymosin β4

The archetypical β-thymosin is β4 (product in humans of the TMSB4X gene), which is a major cellular constituent in many tissues. Its intracellular concentration may reach as high as 0.5 mM. Following Thymosin α1, β4 was the second of the biologically active peptides from Thymosin Fraction 5 to be completely sequenced and synthesized. Any concepts of the biological role of thymosin β4 must inevitably be coloured by the demonstration that total ablation of the thymosin β4 gene in the mouse allows apparently normal embryonic development of mice which are fertile as adults.

Actin binding

Thymosin β4 was initially perceived as a thymic hormone. However this changed when it was discovered that it forms a 1:1 complex with G (globular) actin, and is present at high concentration in a wide range of mammalian cell types. When appropriate, G-actin monomers polymerize to form F (filamentous) actin, which, together with other proteins that bind to actin, comprise cellular microfilaments. Formation by G-actin of the complex with β-thymosin (= "sequestration") opposes this. Due to its profusion in the cytosol and its ability to bind G-actin but not F-actin, thymosin β4 is regarded as the principal actin-sequestering protein in many cell types. Thymosin β4 functions like a buffer for monomeric actin as represented in the following reaction:

Factin ↔ Gactin + Thymosinβ4 ↔ Gactin:Thymosinβ4

Release of G-actin monomers from thymosin β4 occurs as part of the mechanism that drives actin polymerization in the normal function of the cytoskeleton in cell morphology and cell motility. The sequence LKKTET, which starts at residue 17 of the 43-amino acid sequence of thymosin beta-4, and is strongly conserved between all β-thymosins, together with a similar sequence in WH2 domains, is frequently referred to as "the actin-binding motif" of these proteins, although modelling based on X-ray crystallography has shown that essentially the entire length of the β-thymosin sequence interacts with actin in the actin-thymosin complex.


Local protein synthesis plays an essential role in the regulation of various aspects of axonal and dendritic function in adult neurons. At present, however, there is no direct evidence that local protein translation is functionally contributing to neuronal outgrowth. Here, we identified the mRNA encoding the actin-binding protein β-thymosin as one of the most abundant transcripts in neurites of outgrowing neurons in culture. β-Thymosin mRNA is not evenly distributed in neurites, but appears to accumulate at distinct sites such as turning points and growth cones. Using double-stranded RNA knockdown, we show that reducing β-thymosin mRNA levels results in a significant increase in neurite outgrowth, both in neurites of intact cells and in isolated neurites. Together, our data demonstrate that local synthesis of β-thymosin is functionally involved in regulating neuronal outgrowth.

The broad aim of this work was to explore the feasibility of using light-directed perturbation techniques to study cell locomotion. Specifically, a caged form of thymosin β4 (Tβ4) was photoactivated in a defined local region of locomoting fish scale keratocytes and the resulting perturbation of locomotion was studied. Purified Tβ4 was produced in an inactive form by “caging” with ([n-nitroveratryl]oxy)chlorocarbamate. In vitro spectrophotofluorometric assays indicated that caged Tβ4 did not change the normal actin polymerization kinetics, whereas photoactivated Tβ4 significantly inhibited actin polymerization. With an a priori knowledge of the cytoplasmic diffusion coefficient of Tβ4 as measured by fluorescence recovery after photobleaching experiments, the rapid sequestration of actin monomers by uncaged Tβ4 and the consequent reduction in the diffusional spread of the Tβ4–actin complex were predicted using Virtual Cell software (developed at the Center for Biomedical Imaging Technology, University of Connecticut Health Center). These simulations demonstrated that locally photoactivating Tβ4 in keratocytes could potentially elicit a regional locomotory response. Indeed, when caged Tβ4 was locally photoactivated at the wings of locomoting keratocytes, specific turning about the irradiated region was observed, whereas various controls were negative. Additionally, loading of exogenous Tβ4 into both keratocytes and fibroblasts caused very rapid disassembly of actin filaments and reduction of cellular contractility. Based on these results, a mechanical model is proposed for the turning behavior of keratocytes in response to photoreleased Tβ4.

Honkura, Matsuzaki, Noguchi, Ellis-Davies, Kasai • 2008 • Cell • FullText

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SI video1
Movie S7. Two-photon imaging of the confinement of PAGFP-actin fluorescence after repetitive photoactivation and uncaging of MNI-glutamate (60 pulses of 0.6 ms duration at 1 Hz) at a point (square) distal to the apex of the spine shown in Figure 5D. The 2D images were acquired every 10 s for 7 min. Photoactivation was induced at the moment when the white square turns red. Scale bar, 1 μm.

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SI video1
Movie S8. Two-photon imaging of PAGFP-actin fluorescence after repetitive photoactivation and uncaging of MNI-glutamate (60 pulses of 0.6 ms duration at 1 Hz) at a point (square) distal to the apex of the spine shown in Figure 5G in the presence of Lat A (0.1 μM). The 2D images were acquired every 15 s for 10 min. Photoactivation was induced at the moment when the white square turns red. Scale bar, 1 μm.

Fischer, Kaech, Knutti, Matus • 1998 • Cell • FullText

Hover mouse over icon to expand supplementary movies
  • SI video1
    Visualization of actin dynamics in migrating fibroblasts. A rat embryo fibroblast transiently transfected with EGFP was recorded crawling from top right to bottom left of the frame. In addition to the actin dynamics associated with membrane ruffles, note the reorientation of stress fibers as the cell turns slightly toward the left from its initial direction. Time marker shows hours, minutes, and seconds.
  • SI video2
    Growth cone on a 48-hr-old hippocampal neuron transfected with GFP-actin. In addition to the concentration of actin in the “palm” of the growth cone, local concentrations are associated with spots and lateral filopodia from the shaft of the neurite. Time marker shows hours, minutes, and seconds.
  • SI video3
    A large field from a GFP-actin expressing hippocampal neuron is shown at low magnification (40X objective lens). As well as showing the widespread nature of spine motility, this sequence also indicates the extent to which actin is concentrated in spines compared to the shaft domain of dendrites. Time marker shows hours, minutes, and seconds.
  • SI video4
    Dynamics in motile spines recorded at higher magnification (100X objective lens) from a GFP-actin transfected cell in another culture. Over these short recording times the actin-driven changes are limited to spine shape and involve the growth and shrinkage of miniature protrusions. Time marker shows hours, minutes, and seconds.
  • SI video5
    Even over the brief duration of this recording (90 s) in a GFP-actin transfected neuron, continuous changes in the configuration of spine actin are visible. Time marker shows minutes and seconds.
  • SI video6
    The cell shown in Figure 2a was recorded during an experiment to examine the susceptibility of spine motility to the actin polymerization inhibitor cytochalasin D. At the point indicated, medium containing the drug flowed into the observation chamber. Time marker shows hours, minutes, and seconds.

We find that induction of long-term potentiation (LTP) of synaptic transmission in acute hippocampal slices of adult mice evokes a reliable, transient expansion in spines that are synaptically activated, as determined with calcium imaging. Similar to LTP, transient spine expansion requires N-methyl- D-aspartate (NMDA) receptor-mediated Ca2 influx and actin polymerization. Moreover, like the early phase of LTP induced by the stimulation protocol, spine expansion does not require Ca2 influx through L-type voltage-gated Ca2 channels nor does it require protein synthesis. Thus, transient spine expansion is a characteristic feature of the initial phases of plasticity at mature synapses and so may contribute to synapse remodeling important for LTP.

Hover mouse over icon to expand supplementary movies
  • video1
    Typical transient expansion.
    Movie of a spine exhibiting typical transient expansion corresponding to Fig. 1B
    (12 frames acquired every 0.5 min, viewed at 4 frames/s).
    The appearance of the red circle represents stimulation with a 1-s, 100-Hz tetanus.
  • video2
    Movie 2. Asymmetric transient expansion.
    Movie of a spine exhibiting asymmetric transient expansion
    corresponding to Fig. 1D (12 frames acquired every 0.5 min, viewed at 4 frames/s).
    The appearance of the red circle represents stimulation with a 1-s, 100-Hz tetanus.
  • video3
    Movie 3. Transient spine expansion on large- and small-diameter dendritic branches.
    Movie of three dendritic branches with shafts of varying diameter from a single
    CA1 pyramidal neuron exhibiting many spines undergoing transient expansion
    (12 frames acquired every 0.5 min, viewed at 4 frames/s).
    The appearance of the red circle represents stimulation with a 1-s, 100-Hz tetanus.

Hover mouse over icon to expand supplementary movies
  • SI movie1
    SI Movie 1. Time lapse (230´) movie of the elongation of actin filaments by 3 mM Mg-ADP-actin
    (30% Alexa-Green label) in polymerization buffer with 0.2 mM ADP,
    viewed in a flow chamber coated with 50 nM NEM-myosin and 1% BSA.
  • SI movie2
    Time lapse (225) movie of the elongation and depolymerization of ADP-actin filaments (30% Alexa label).
    The movie begins with filaments elongating in 5 mM Mg-ADP-actin in polymerization buffer.
    At the point indicated by WASHOUT, free actin monomers were washed out of the
    chamber with polymerization buffer with 0.2 mM ADP to allow depolymerization.

Koskinen and Hotulainen • 2014 • Frontiers • FullText

Measurements of actin turnover in dendritic spines: Fitting the data from individual measurements resulted in a mean stable component size of 18% as well as mean time constants of 51 sec for the dynamic component and 840 sec for the stable component.

Bindschadler, Osborn, Dewey, McGrath • 2004 • BiophysicalJ • PMC

Actin polymerization proceeds until only a small concentration (~0.1 µM) of unpolymerized actin (Gactin) remains. This ‘‘critical concentration’’ is also the minimum concentration required to form filaments (F-actin).

Both regulated and unregulated actin binding proteins modify the actin cycle in cells (Fig. 1). Barbed-end binding proteins block the assembly of G-actin at filament-barbed ends. The most abundant barbed-end binding proteins, capping protein (CP) and gelsolin (Isenberg et al., 1980; Yin et al., 1981), are inactivated by PIP2 and other polyphosphoinositides (Heiss and Cooper, 1991; Janmey and Stossel, 1987). Gelsolin, which also severs actin filaments (Yin and Stossel, 1979), requires micromolar calcium for its activity. CP, gelsolin, and Arp2/3 complex (Mullins et al., 1998), can nucleate new actin filaments. The processes of severing and nucleation help determine the number and length of actin filaments. Arp2/3 complex can also cap pointed ends (Mullins et al., 1998). Arp2/ 3 complex activities are greatly enhanced by the GTPase binding protein N-WASp (Machesky et al., 1999; Yarar et al., 1999). Inhibited by phosphorylation (Morgan et al., 1993), the ADF/cofilin family proteins bind preferentially to ADP containing subunits (Carlier et al., 1997). Cofilin destabilizes filaments by severing them (Maciver et al., 1991), by accelerating the rate of ADP subunit disassembly (Carlier et al., 1997), and by enhancing the rate of Pi release (Blanchoin and Pollard, 1999). Unregulated proteins of the b4-thymosin family bind actin monomers to maintain unpolymerized actin at hundreds of times the critical concentration (Safer et al., 1990). Unlike b4-thymosin, the monomer binding protein profilin has catalytic functions. Profilin accelerates the exchange of ADP for ATP on actin monomer 140-fold (Selden et al., 1999). Also unlike actin complexed with b4-thymosin, profilin-bound G-actin assembles at barbed ends but not pointed ends (Pollard and Cooper, 1984), releasing unbound profilin (Pantaloni and Carlier, 1993).